Susceptibility locus for schizophrenia

ABSTRACT

Provided are methods for determining the susceptibility of an individual to a neuropsychiatric disorder, or a method of diagnosis or prognosis of the neuropsychiatric disorder (particularly schizophrenia) the methods comprising use of a pericentriolar material 1 (PCM1) marker which is located in the chromosomal region 8p21-22, for example a marker within the PCM1 gene locus or within 1000 kb of it. The invention also provides novel markers, and related materials and methods of detecting them, and identifying further molecules for use in therapy and diagnosis.

TECHNICAL FIELD

This invention relates to the identification of chromosomal regions onchromosome 8 linked to genetic sequences which affect susceptibility toschizophrenia.

BACKGROUND OF INVENTION

The schizophrenias are a collection of psychotic psychiatric disorderswith a lifetime prevalence of approximately 0.85% in the generalpopulation. It is one of the most prevalent and potentially devastatingof the neuropsychiatric disorders and is characterised by episodes ofauditory hallucinations, delusions, thought disorder, inappropriateaffect, bizarre behaviour and cognitive abnormalities.

Currently, individuals are typically evaluated for schizophrenia usingthe criteria set forth in the most current version of the InternationalClassification of diseases or the American Psychiatric Association'sDiagnostic and Statistical Manual of Mental Disorders (DSM). Some of theavailable drug treatments are effective in only approximately a third ofindividuals diagnosed with schizophrenia, a third respond partially anda third respond poorly. It has not so far been possible to predict whichdrug treatments will be effective in a particular schizophrenia patient.

The existence of a genetic component for schizophrenia is supported bytwin studies and genetic linkage studies (Gurling, 1996, The Genetics ofthe Schizophrenias, In: Genetics of Mental Disorders Part II: ClinicalIssues, Eds: Papadimitriou, G. N., Mendlewicz, J. Balliere's ClinicalPsychiatry, International Practice and Research 2:15-46).

Mapping genes for common diseases such as schizophrenia is complicatedby the variable definition of schizophrenia phenotypes, by aetiologicheterogeneity, and by uncertainty about the mode of genetic transmissionof the disease trait. With neuropsychiatric disorders there is ambiguityin distinguishing individuals who carry the affected genotype from thosewho are genetically unaffected.

Thus, one of the greatest difficulties facing psychiatric geneticists isuncertainty regarding the validity of phenotype designations, sinceclinical diagnoses are based solely on clinical observation andsubjective reports.

DISCLOSURE OF THE INVENTION

The present inventors have identified a region on chromosome 8 thatharbours a susceptibility locus for schizophrenia. In particular, theyhave identified a gene (PCM1) which appears to have a role inschizophrenia.

Previously, linkage studies have indicated that a region of chromosome 8(8p21-22) is linked to schizophrenia (Kendler, et al 1996; Levinson etal.,1996). For example, a marker D8S261 was shown to be located in thisregion. However, the results from such linkage analysis are only able tolocalise the disease to around 30 million to 60 million base pairs. Suchlinkage results span a region which is far too broad to represent asingle gene. Moreover, it is not predictable from such linkage analysiswhere in the large region, the schizophrenia susceptibility gene lies,nor to be able to identify a single gene by positional cloning orcandidate gene analysis within such a large region.

The present inventors employed linkage disequilibrium mapping andallelic association studies to identify the region of chromosome 8p21-22which is involved in schizophrenia.

In the first stage, the inventors searched for di, tri and tetra CA/GTnucleotide repeat motifs and identified seven novel polymorphisms, ormicrosatellite marker loci, (D8S2612; D8S2613; D8S2614; D8S2615;D8S2616; D8S2617; D8S2618). These all localised to chromosome 8p21.3.Allelic association with schizophrenia was demonstrated for two of these(D8S2615; D8S2616) in studies with 137 schizophrenia patients and 300ethnically matched controls, in a UK population.

In addition, the inventors demonstrated strong allelic association witha known microsatellite marker (D8S261).

These markers were shown by the inventors to be localised to thechromosome region 8p21.3. D8S261, D8S2615 and D8S2616 were localised tothe PCM1 gene

D8S2612; D8S2613; D8S2614; D8S2617; D8S2618 were localised as being lessthan one megabase from the first marker which was shown to be in allelicassociation with DS261.

The present inventors then searched for polymorphisms within the PCM1gene. After identifying the intron-exon boundaries, they carried outautomated bi-directional sequencing of PCR-amplified exons and of 100 bpof intron sequence either side of the exons. This analysis was carriedout on 19 schizophrenia cases from the sample used in the associationstudy, 26 cases from British multiply-affected schizophrenia families,and 10 healthy controls. In order to increase the chances of detecting apolymorphism in linkage disequilibrium with schizophrenia, affectedindividuals who carried the polymorphism in D8S2616, D8S2615, or D8S261associated with schizophrenia were selected.

The inventors screened exons 4 and 5 because of their finding thatD82616, which is located at the boundary of these two exons, has strongallelic association with schizophrenia.

As a result, the inventors identified single nucleotide polymorphisms inthe following locations: position 80254 in the intronic sequence 3′ toexon 4; position 80123 in exon 4; position 87366 in the intronicsequence 5′ of exon 5; position 87507 in the intronic sequence 5′ toexon 5.

These results have major implications for methods of treatment, methodsof diagnosis or prognosis, methods of identifying compounds for use inmethods of treatment, prognosis or diagnosis, providing compounds foruse in such methods of treatment, prognosis or diagnosis.

For the purposes of this application, the various aspects are discussedwith particular reference to the PCM1 gene. However, it would be clearto the skilled person that, should another gene within the regionidentified by the inventors be found to be a relevant gene, that thevarious aspects of the invention would apply equally to that gene.

Where the term PCM1 marker is used this is taken as meaning a markerwithin an intron or exon of the PCM1 gene, or in which is in allelicassociation or linkage disequilibrium with the PCM1 gene.

The inventor's have found the region surrounding the PCM1 gene to have alow evolutionary rate of recombination. Therefore the distance overwhich linkage disequilibrium may be found is relatively high.Accordingly a PCM1 polymorphic marker may be within 3000 kb (eitherside) of the PCM1 gene, preferably within 1000 kb of the PCM1 gene, morepreferably within 500 kb of the PCM1 gene, more preferably within 100 kbof the PCM1 gene, more preferably within 50 kb of the PCM1 gene, mostpreferably within 10 kb of the PCM1 gene.

In a first aspect of the present invention there is disclosed a methodfor assessing the susceptibility of an individual for schizophrenia, ora method of diagnosis or prognosis of schizophrenia, which methodcomprises (i) obtaining a sample of genomic DNA from an individual(which sample may be all or part of the material to be analysed), (ii)using a PCM1 marker located in the chromosomal region 8p21.3 to assessthe susceptibility or make said diagnosis or prognosis.

The preferred PCM1 markers may be allelic or other polymorphic markersas defined above. Preferably these markers are within the PCM1 gene.

Preferred PCM1 markers are D8S2612; D8S2613; D8S2614; D8S2615; D8S2616;D8S2617; D8S2618, D8S261. More preferred are D8S2615 and D8S2616, andD8S261, most preferred are D8S2615 and D8S2616. The location of thesemarkers is described elsewhere herein.

It is to be noted that, while allelic association was shown by thepresent inventors for D8S2615 D8S2616 and D8S261 in a UK population,these and the remaining PCM1 markers; D8S2612, D8S2613, D8S2614,D8S2617, and D8S2618 may also provide useful diagnostic markers in other(for example, non-UK) populations.

Alternatively, or additionally single nucleotide polymorphisms (SNP), asdescribed herein, may be used as markers. These include: the SNP atposition 80254 of the AB020866 genomic clone; the SNP at position 80123of the AB020866 genomic clone; the SNP at position 87366 of the AB020866genomic clone; and the SNP at position 87507 of the AB020866 genomicclone. Further details of these SNPs are given elsewhere herein.

In further aspects, the present invention discloses the use of the PCM1gene, or the polypeptide encoded by the PCM1 gene, in a method foridentifying the susceptibility of an individual for schizophrenia, or ina method of diagnosis or prognosis of schizophrenia.

The PCM1 gene has been sequenced and the wild-type coding and regulatorysequences can be found in clones AB020866 and AB020867 of the contigNT_(—)000501 [GenomeChannel:http//genome.ornl.gov/GC/cgi-bin/GCKSearchForm.cgi]. Forreference, the mRNA sequence of the longest open reading frame is shownin FIG. 1 for the wild type.

Sequence for the 8p21.3 region is published in the ENTREZ database inclone AB020866 and flanking clones.

Where the various aspects of the invention use, or involve, apolypeptide encoded by the PCM1 gene, it is preferred that thepolypeptide is that shown in FIG. 1.

In a further aspect the present invention discloses a method foridentifying the susceptibility of an individual for schizophrenia, or amethod of diagnosis or prognosis of schizophrenia, wherein said methodcomprises: obtaining a nucleic acid sample from an individual; anddetermining in that sample, the presence or absence of mutations orpolymorphisms in the PCM1 gene.

A corresponding method may comprise: obtaining a nucleic acid or proteinsample from an individual; and determining the level of expression fromthe PCM1 gene.

Where a nucleic acid sample is used, the level of expression may bedetermined by measuring the amount of mRNA. Where a protein sample isused, the level of PCM1 gene expression may be determined by measuringthe amount of PCM1 polypeptide product.

Another aspect of the present invention is a method for identifying orisolating genetic loci associated with susceptibility to schizophreniacomprising screening genomic libraries with genetic sequence derivedfrom PCM1 polymorphic markers located in the chromosomal region 8p21.3and identifying open reading frames in regions adjacent to said geneticsequence. The preferred markers are those indicated above.

A region which is described as ‘adjacent’ to a genetic sequence may bewithin about 3000 kb of the marker, preferably within about 1000 kb,within about 500 kb away, and more preferably within about 100 kb, morepreferably within 50 kb, more preferably within 10 kb of the marker.

For example, an open reading frame which is adjacent the PCM1 gene maybe within about 3000 kb of the marker (since the recombination frequencyis quite low, as discussed earlier), preferably within about 1000 kb,within about 500 kb away, and more preferably within about 100 kb, morepreferably within 50 kb, more preferably within 10 kb of the marker.

Another aspect of the present invention is a method for mapping lociwhich affect susceptibility to schizophrenia by comparing a genomicregion containing a particular allele of a PCM1 polymorphic markerlocated in the chromosomal region 8p21.3 with a genomic regioncontaining a different allele of the same marker. The preferred markersare those indicated above.

Where the present invention relates to the analysis of nucleic acid orprotein of an individual, such an individual may be one who hasschizophrenia, is considered at risk from schizophrenia (e.g. by havinga sibling with and/or family history of schizophrenia), or may besymptomless. The sample from the individual may be prepared from anyconvenient sample, for example from blood or skin tissue. A sampleobtained from an individual may be analysed according to methods of thepresent invention. Methods of the present invention may thereforeinclude providing a sample of nucleic acid, or protein obtained from anindividual.

In a further aspect the invention provides a nucleic acid moleculecomprising the PCM1 gene for use in the treatment of schizophrenia.

In a further aspect the invention provides the use of the PCM1 gene inthe manufacture of a medicament for the treatment of schizophrenia.

In a further aspect the invention provides a method of treatment forschizophrenia comprising administering to a patient a nucleic acidmolecule having the sequence of the PCM1 gene. The nucleic acid moleculemay be within a cell, and the cell containing the nucleic acid moleculemay be administered to the patient.

In a further aspect the invention provides the polypeptide encoded bythe PCM1 gene for use in the treatment of schizophrenia.

In a further aspect the invention provides the use of a polypeptideencoded by the PCM1 gene in the manufacture of a medicament for thetreatment of schizophrenia.

In a further aspect the invention provides a method of treatment forschizophrenia comprising administering to a patient a polypeptideencoded by the nucleic acid molecule having the sequence of the PCM1gene.

In a further aspect the invention provides a method of treatment ofschizophrenia comprising administering to a patient a substance whichmodulates expression from the PCM1 gene, or administering a compoundwhich modulates the level of activity of the PCM1 gene product.

In a further aspect the invention provides a method of identifying amolecule for use in the diagnosis, prognosis or treatment ofschizophrenia.

Accordingly, a method of identifying a molecule for use in thediagnosis, prognosis or treatment of schizophrenia may comprise:

-   -   admixing a test substance with a nucleic acid molecule        comprising the PCM1 gene; and measuring the level of expression        from that nucleic acid molecule. As discussed in more detail        later, such a method is typically performed using an expression        system which includes a nucleic acid molecule comprising the        PCM1 gene. The level of expression can be measured by measuring        the amount of PCM1 mRNA or PCM1 polypeptide product, or of a        reporter gene which is linked to the PCM1 gene (or portion        thereof, e.g promoter) in the expression system.

For example, for identification of molecules for treatment, therapeuticmolecules may be identified, these molecules may interact with PCM1 ormolecules that interact with other proteins that interact with PCM1,such that expression of PCM1 containing the schizophrenia associatedpolymorphism is reduced or so that proteins which counter its effect areincreased in expression. For example, after expression oroverexpression, protein isolation and crystallisation of the PCM1protein can be achieved which will permit the identification of proteinfolds and 3D structure with X rays. Furthermore, by inference from thetypes of amino acid residues and their position in the PCM1 proteinspecific target regions of the protein may be identified as sites wherenew treatments for schizophrenia can be targeted.

Other methods include the “Yeast two hybrid system” and the use oftransgenic mutant PCM1 animals, conditional (CRE/LOX) mutants orknockout animal models and then using the absence or relative absence orover expression of PCM1 to develop a new pharmaceutical agent such as asmall molecule, peptide, oligonucleotide or antibody so that theexpression of too much, too little or of abnormal mutant PCM1 protein isameliorated. Further, the expression of PCM1 protein in cells grown inan in vivo, ex vivo or in vitro method enables therapeutic drugcandidate molecules to be tested to detect an effect on the expressionof normal and abnormal mutant PCM1 proteins and proteins that interactwith PCM1 so that a new therapeutic agent can be created. Furtherdescription of such methods is given later.

In a further aspect the invention provides a method of identifying amolecule for use in the diagnosis, prognosis or treatment ofschizophrenia, which method comprises:

-   -   admixing a test substance with a polypeptide encoded by a        nucleic acid molecule comprising the PCM1 gene; and measuring        the level of activity of the polypeptide. Such a method may be        carried out using a sample of isolated polypeptide, or may be        carried out using a nucleic acid molecule comprising the PCM1        gene which is contained within an expression system, such that        the polypeptide is expressed from the nucleic acid.

In a further aspect the invention provides a method of identifying amolecule for use in the diagnosis, prognosis or treatment ofschizophrenia, which method comprises:

-   -   admixing a test substance with a nucleic acid molecule        comprising the PCM1 gene, or with a polypeptide encoded by a        nucleic acid molecule comprising the PCM1 gene; and determining        the binding of the test substance to the polypeptide. Such a        method may be carried out using a nucleic acid molecule        comprising the PCM1 gene which is contained within an expression        system.

Further methods of identifying molecules for use in a method ofdiagnosis or prognosis of schizophrenia include methods of identifyingsubstances which bind to the PCM1 nucleic acid sequence or to the PCM1polypeptide product, and methods of identifying substances which inhibitthe interaction of the PCM1 gene or PCM1 gene product with its “bindingpartners”.

In any of the above methods, the results may be compared with resultsfrom a control sample, in which no test substance was included.

Substances identified by the above methods (which may include novelsubstances) therefore represent further aspects of the invention.

In further aspects the invention provides an isolated nucleic acid whichis a polymorphic variant of the nucleic acid sequence having thesequence shown in FIG. 1, such a sequence may have the polymorphicvariation shown in any one of FIG. 2, 3, 4 or 5, or a combination ofthese. The invention further provides polypeptides encoded by thesesequences where these differ from that encoded by FIG. 1. These nucleicacids have been shown by the inventors to have a role in schizophrenia.

It should be noted that The chromosome 8p21-22 PCM1 subtype ofschizophrenia can also manifest itself as other psychiatric disorders.For example Kendler et al 1988 (Kendler, K. S., Gruenberg, A. M. &Tsuang, M. T. (1988) A family study of the subtypes of schizophrenia. AmJ Psychiatry, 145, 57-62.) found that different subtypes ofschizophrenia, schizoaffective disorder and schizotypal disorderappeared to share the same genetic aetiology within families whereschizophrenia is present. In addition subtypes of common psychiatricdisorders can also share the same genetic aetiology as schizophrenia asshown by Tsuang et al 1991 (Tsuang, M. T. (1991) Morbidity risks ofschizophrenia and affective disorders among first-degree relatives ofpatients with schizoaffective disorders. Br J Psychiatry, 158,165-170.8p21-22).

Although uncommon, subtypes of neuroses such as affective disorder,obsessive compulsive disorder, phobias and anxiety states can also becaused by the same genetic aetiology as schizophrenia. These disordersare sometimes diagnosed as “pseudoneurotic schizophrenia”. Thus wherethe term “schizophrenia” is used herein, it will be understood that itencompasses also these related disorders, and that the various aspectsof the invention also apply to other neuropsychiatric disorders relatingto the PCM1 gene, such as the related neuropsychiatric disorders,including delusional disorders, reality distortion syndrome, psychomotorretardation syndrome, confusion syndrome, paraphrenia, paranoidpsychosis, schizotypal disorder, schizoaffective disorder,schizoaffective schizophrenia, psychogenic psychosis, catatonia,periodic schizophrenia, cycloid personality disorder, schizophreniarelated affective disorders and subtypes of unipolar affective disorder,attention deficit disorder and magical thinking.

Various aspects of the invention will now be described in more detail.

Diagnostic Methods

The methods of the invention may use a variety of techniques and for thediagnosis or prognosis of schizophrenia, or for identification ofsubjects having a predisposition to, or susceptibility for,schizophrenia.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one nucleic acidcomprising the PCM1 gene, or the PCM1 polypeptide product, or comprisingan antibody specific for PCM1, which may be used e.g., in clinicalsettings, in a method of diagnosis, or prognosis of schizophrenia.Accordingly, such a kit represents a further aspect of the invention.

Examples of techniques which may be used in the methods of the inventionare described below.

Detection of PCM1 Nucleic Acid Molecules

A variety of methods can be employed to screen for the presence of PCM1mutations and to detect and/or assay levels of PCM1 nucleic acidsequences.

Mutations within the PCM1 gene can be detected by utilizing a number oftechniques. Nucleic acid from any nucleated cell can be used as thestarting point for such assay techniques, and may be isolated accordingto standard nucleic acid preparation procedures that are well known tothose of skill in the art.

PCM1 nucleic acid sequences may be used in hybridization oramplification assays of biological samples to detect abnormalitiesinvolving PCM1 gene structure, including point mutations, insertions,deletions, inversions, translocations and chromosomal rearrangements.Such assays may include, but are not limited to, Southern analyses,single-stranded conformational polymorphism analyses (SSCP), and PCRanalyses.

Mutations and polymorphisms in the gene may be detected using DNA (orRNA) sequence analysis, using techniques which are standard in the art.Examples of suitable techniques may be found in Sambrook et al., 1987.

Diagnostic methods for the detection of PCM1 gene-specific mutations caninvolve for example, contacting and incubating nucleic acids includingrecombinant DNA molecules, cloned genes or degenerate variants thereof,obtained from a sample, e.g., derived from a patient sample or otherappropriate cellular source, with one or more labeled nucleic acidreagents including recombinant DNA molecules, cloned genes or degeneratevariants thereof, as described elsewhere herein, under conditionsfavorable for the specific annealing of these reagents to theircomplementary sequences within the PCM1 gene. Preferably, the lengths ofthese nucleic acid reagents are at least 15 to 30 nucleotides.

After incubation, all non-annealed nucleic acids are removed from thenucleic acid:PCM1 molecule hybrid. The presence of nucleic acids thathave hybridized, if any such molecules exist, is then detected. Usingsuch a detection scheme, the nucleic acid from the cell type or tissueof interest can be immobilized, for example, to a solid support such asa membrane, or a plastic surface such as that on a microtitre plate orpolystyrene beads. In this case, after incubation, non-annealed, labelednucleic acid reagents are easily removed. Detection of the remaining,annealed, labeled PCM1 nucleic acid reagents is accomplished usingstandard techniques well-known to those in the art. The PCM1 genesequences to which the nucleic acid reagents have annealed can becompared to the annealing pattern expected from a normal PCM1 genesequence in order to determine whether a PCM1 gene mutation is present.

Alternative diagnostic methods for the detection of PCM1 gene specificnucleic acid molecules, in patient samples or other appropriate cellsources, may involve their amplification, e.g., by PCR (the experimentalembodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), followedby the detection of the amplified molecules using techniques well knownto those of skill in the art. The resulting amplified sequences can becompared to those that would be expected if the nucleic acid beingamplified contained only normal copies of the PCM1 gene in order todetermine whether a PCM1 gene mutation exists.

Additionally, well-known genotyping techniques can be performed toidentify individuals carrying PCM1 gene mutations. Such techniquesinclude, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one of therecognition sites for the specific restriction enzyme used.

Additionally, improved methods for analyzing DNA polymorphisms, whichcan be utilized for the identification of PCM1 gene mutations, have beendescribed that capitalize on the presence of variable numbers of short,tandemly repeated DNA sequences between the restriction enzyme sites.For example, Weber (U.S. Pat. No. 5,075,217) describes a DNA markerbased on length polymorphisms in blocks of (dC-dA)n-(dG-dT)n shorttandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks isestimated to be 30,000-60,000 bp. Markers that are so closely spacedexhibit a high frequency co-inheritance, and are extremely useful in theidentification of genetic mutations, such as, for example, mutationswithin the PCM1 gene, and the diagnosis of diseases and disordersrelated to PCM1 mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759) describe a DNA profilingassay for detecting short tri and tetra nucleotide repeat sequences. Theprocess includes extracting the DNA of interest, such as the PCM1 gene,amplifying the extracted DNA, and labelling the repeat sequences to forma genotypic map of the individual's DNA.

The level of PCM1 gene expression may be assayed. For example, RNA froma cell type or tissue known, or suspected, to express the PCM1 gene,such as brain, may be isolated and tested utilizing hybridization or PCRtechniques such as are described, above. The isolated cells can bederived from cell culture or from a patient. The analysis of cells takenfrom culture may be a necessary step in the assessment of cells to beused as part of a cell-based gene therapy technique or, alternatively,to test the effect of compounds on the expression of the PCM1 gene. Suchanalyses may reveal both quantitative and qualitative aspects of theexpression pattern of the PCM1 gene, including activation orinactivation of PCM1 gene expression.

In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). A sequence within the cDNAis then used as the template for a nucleic acid amplification reaction,such as a PCR amplification reaction, or the like. The nucleic acidreagents used as synthesis initiation reagents (e.g., primers) in thereverse transcription and nucleic acid amplification steps of thismethod are chosen from among the PCM1 gene nucleic acid reagentsdescribed herein. The preferred lengths of such nucleic acid reagentsare at least 9-40 nucleotides, preferably 15 to 30, more preferably19-28 nucleotides in length.

For detection of the amplified product, the nucleic acid amplificationmay be performed using radioactively or non-radioactively labelednucleotides. Alternatively, enough amplified product may be made suchthat the product may be visualized by standard ethidium bromide stainingor by utilizing any other suitable nucleic acid staining method.

Additionally, it is possible to perform such PCM1 gene expression assays“in situ”, i.e., directly upon tissue sections (fixed and/or frozen) ofpatient tissue obtained from biopsies or resections, such that nonucleic acid purification is necessary. Nucleic acid reagents such asthose described herein may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, “PCR In SituHybridization: Protocols And Applications”, Raven Press, NY).

Alternatively, if a sufficient quantity of the appropriate cells can beobtained, standard Northern analysis can be performed to determine thelevel of mRNA expression of the PCM1 gene.

(2) Detection of PCM1 Gene Products

Antibodies directed against unimpaired or mutant PCM1 gene products orconserved variants or peptide fragments thereof, which are discussed, asdiscussed elsewhere herein, may also be used as diagnostics andprognostics for schizophrenia. Such methods may be used to detectabnormalities in the level of PCM1 gene product synthesis or expression,or abnormalities in the structure, temporal expression, and/or physicallocation of PCM1 gene product. Accordingly, the use of such antibodiesin methods of diagnosis or prognosis of schizophrenia, and methods ofdiagnosis or prognosis which use these antibodies represent furtheraspects of the invention.

The antibodies and immunoassay methods described below have, forexample, important in vitro applications in assessing the efficacy oftreatments for PCM1 disorders or neuropsychiatric disorders, such asschizophrenia. Antibodies, or fragments of antibodies, such as thosedescribed below, may be used to screen potentially therapeutic compoundsin vitro to determine their effects on PCM1 gene expression and PCM1peptide production. The compounds that have beneficial effects on anPCM1 disorder or a neuropsychiatric disorder, such as schizophrenia, canbe identified, and a therapeutically effective dose determined.

In vitro immunoassays may also be used, for example, to assess theefficacy of cell-based gene therapy for schizophrenia. Antibodiesdirected against PCM1 peptides may be used in vitro to determine, forexample, the level of PCM1 gene expression achieved in cells geneticallyengineered to produce PCM1 peptides. In the case of intracellular PCM1gene products, such an assessment is done, preferably, using celllysates or extracts. Such analysis will allow for a determination of thenumber of transformed cells necessary to achieve therapeutic efficacy invivo, as well as optimization of the gene replacement protocol.

The tissue or cell type to be analyzed will generally include those thatare known, or suspected, to express the PCM1 gene. The protein isolationmethods employed herein may, for example, be such as those described inHarlow and Lane (1988, “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). The isolated cellscan be derived from cell culture or from a patient. The analysis ofcells taken from culture may be a necessary step in the assessment ofcells to be used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of thePCM1 gene.

Preferred diagnostic methods for the detection of PCM1 gene products(and therefore of diagnosis or prognosis of schizophrenia) or conservedvariants or peptide fragments thereof, include immunoassays wherein thePCM1 gene products or conserved variants or peptide fragments aredetected by their interaction with an anti-PCM1 gene product-specificantibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed herein, useful in the present invention may be used toquantitatively or qualitatively detect the presence of PCM1 geneproducts or conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below,) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection. Such techniquesare especially preferred for PCM1 gene products that are expressed onthe cell surface.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of PCM1 geneproducts or conserved variants or peptide fragments thereof. In situdetection may be accomplished by removing a histological specimen from apatient, and applying thereto a labelled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the PCM1 gene product, or conserved variants orpeptide fragments, but also its distribution in the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of a wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

Immunoassays for PCM1 gene products or conserved variants or peptidefragments thereof will typically comprise incubating a sample, such as abiological fluid, a tissue extract, freshly harvested cells, or lysatesof cells, that have been incubated in cell culture, in the presence of adetectably labeled antibody capable of identifying PCM1 gene products orconserved variants or peptide fragments thereof, and detecting the boundantibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled PCM1 gene specificantibody. The solid phase support may then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label onsolid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-PCM1 gene product antibodymay be determined according to well known methods. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

One of the ways in which the PCM1 gene peptide—specific antibody can bedetectably labeled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)”, 1978, Diagnostic Horizons 2, 1-7, MicrobiologicalAssocia'es Quarterly Publication, Walkersville, Md.); Voller, A. et al.,1978, J. Clin. Pathol. 31, 507-520; Butler, J. E., 1981, Meth. Enzymol.73, 482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, BocaRaton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay,Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety that can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Enzymes that can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect PCM1 gene peptides throughthe use of a radioimmunoassay (RIA) (see, for example, Weintraub, B.,Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques, The Endocrine Society, March, 1986). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase, green fluorescent protein andaequorin.

The diagnostic and prognostic methods of the invention may be performedon a historical sample, and the determination of the presence or absenceof mutations, or the determination of the alleles of the polymorphismsdescribed herein may be performed by analyzing DNA sequences which arestored on a database. Such a diagnostic method of the invention maycomprise searching, for example a databank for a mutation within thePCM1 gene by comparison of the PCM1 gene with the sequence of thewild-type PCM1 gene, or by comparison with one of the PCM1 genesequences shown in the Figures.

Methods to Identify Compounds that Modulate PCM1 Gene Activity

The following assays may be used in the methods of the invention whichinvolve to the identification of molecules which modulate the expressionfrom the PCM1 gene, or modulate the activity of the PCM1 gene product,or that bind to a PCM1 gene product, or that interfere with theinteraction of a PCM1 gene product with intracellular proteins (“bindingpartners”). Therefore, methods of identifying a compound for use in thediagnosis, prognosis or treatment of schizophrenia which may be based onthe following assays, represent further aspects of the invention.

Assays may be utilized that identify compounds that bind to PCM1 generegulatory sequences (e.g., promoter sequences; see e.g., Platt, 1994,J. Biol. Chem. 269, 28558-28562), and that may modulate the level ofPCM1 gene expression. Compounds may include, but are not limited to,small organic molecules, such as ones that are able to cross theblood-brain barrier, gain entry into an appropriate cell and affectexpression of the PCM1 gene or some other gene involved in a PCM1regulatory pathway, or intracellular proteins.

Methods for the identification of such intracellular proteins aredescribed, below. Such intracellular proteins may be involved in thecontrol and/or regulation of mood, or may affect the level of PCM1 geneexpression and/or PCM1 gene product activity. Such compounds may be usedin the therapeutic treatment schizophrenia as described below.

Compounds identified by such methods (which include novel compounds)represent further aspects of the invention. Such compounds may include,but are not limited to, peptides such as, for example, soluble peptides,including but not limited to, Ig-tailed fusion peptides, and members ofrandom peptide libraries; (see, e.g., Lam, et al., 1991, Nature 354,82-84; Houghten, et al., 1991, Nature 354, 84-86), and combinatorialchemistry-derived molecular library made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang, et al., 1993, Cell 72, 767-778), antibodies (including,but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and FAb, F(ab′)₂ and Fab expressionlibrary fragments, and epitope-binding fragments thereof), and smallorganic or inorganic molecules. Such compounds may further comprisecompounds, in particular drugs or members of classes or families ofdrugs, known to ameliorate or exacerbate the symptoms of aneuropsychiatric disorder such as schizophrenia. Such compounds includeantidepressants such as lithium salts, flupenthixol, risperidone,clozapine, quetiapine, olanzapine, haloperidol, droperidol,chlorpromazine, prochloperazine, phenothiazaine derivatives, promazine,trifluopromazine, butyrophenone derivatives, thioxanthine derivativessuch as chlorprothixene and dibenzodiazepines and antipsychoticantiepileptic drugs such carbamazepine, and valproic acid, reserpine.Psychotogenic drugs such as bromocriptine, apomorphine, amphetamine,methylphenidate, methylamphetamine, ketamine, Many of these drugs areused in combination.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of the PCM1gene product, and for ameliorating PCM1 disorders or neuropsychiatricdisorders, such as schizophrenia.

The use of the following methods in the identification of compounds foruse in the diagnosis, prognosis, or treatment of schizophrenia representfurther aspects of the invention.

(1) In vitro Screening Assays for Compounds that Bind to the PCM1 GeneProduct

In vitro systems may be designed to identify compounds capable ofbinding the PCM1 gene products. Compounds identified may be useful, forexample, in modulating the activity of unimpaired and/or mutant PCM1gene products, may be useful in elaborating the biological function ofthe PCM1 gene product, may be utilized in screens for identifyingcompounds that disrupt normal PCM1 gene product interactions, or may inthemselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to thePCM1 gene product may involve preparing a reaction mixture comprisingthe PCM1 gene product and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays may be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringPCM1 gene product or the test substance onto a solid phase and detectingPCM1 gene product/test compound complexes anchored on the solid phase atthe end of the reaction. In one embodiment of such a method, the PCM1gene product may be anchored onto a solid surface, and the testcompound, which is not anchored, may be labeled, either directly orindirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labelled antibody specific for the previously non-immobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody)

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for PCM1 geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

(2) Assays for Intracellular Proteins that Interact with PCM1 GeneProducts

Any method suitable for detecting protein-protein interactions may beemployed for identifying PCM1 protein-protein interactions.

Among the traditional methods that may be employed areco-immunoprecipitation, cross-linking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of proteins, including intracellularproteins, that interact with PCM1 gene products. Once isolated, such aprotein can be identified and can be used in conjunction with standardtechniques, to identify proteins it interacts with. For example, atleast a portion of the amino acid sequence of a protein that interactswith the PCM1 gene product can be ascertained using techniques wellknown to those of skill in the art, such as via the Edman degradationtechnique (see, e.g., Creighton, 1983, “Proteins: Structures andMolecular Principles,” W. H. Freeman & Co., N.Y., pp.34-49). The aminoacid sequence obtained may be used as a guide for the generation ofoligonucleotide mixtures that can be used to screen for gene sequencesencoding such proteins. Screening made be accomplished, for example, bystandard hybridization or PCR techniques. Techniques for the generationof oligonucleotide mixtures and the screening are well-known. (See,e.g., Ausubel, supra, and 1990, “PCR Protocols: A Guide to Methods andApplications,” Innis, et al., eds. Academic Press, Inc., New York).

Additionally, methods may be employed that result in the simultaneousidentification of genes that encode the a protein which interacts withan PCM1 protein. These methods include, for example, probing expressionlibraries with labeled PCM1 protein, using PCM1 protein in a mannersimilar to the well known technique of antibody probing of lambda.gt11and lambda.gt10 libraries.

One method that detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien, etal., 1991, Proc. Natl. Acad. Sci. USA, 88, 9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.)

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to the PCM1 gene product and theother consists of the transcription activator protein's activationdomain fused to an unknown protein that is encoded by a cDNA that hasbeen recombined into this plasmid as part of a cDNA library. TheDNA-binding domain fusion plasmid and the cDNA library are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., HBS or lacZ) whose regulatory region contains thetranscription activator's binding site. Either hybrid protein alonecannot activate transcription of the reporter gene: the DNA-bindingdomain hybrid cannot because it does not provide activation function andthe activation domain hybrid cannot because it cannot localize to theactivator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, PCM1 geneproducts may be used as the bait gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait PCM1 gene productfused to the DNA-binding domain are co-transformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation, abait PCM1 gene sequence, such as the open reading frame of the PCM1gene, can be cloned into a vector such that it is translationally fusedto the DNA encoding the DNA-binding domain of the GAL4 protein. Thesecolonies are purified and the library plasmids responsible for reportergene expression are isolated. DNA sequencing is then used to identifythe proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait PCM1 gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait PCM1 gene-GAL4 fusion plasmid into a yeast strain that containsa lacZ gene driven by a promoter that contains GAL4 activation sequence.A cDNA encoded protein, fused to GAL4 transcriptional activation domain,that interacts with bait PCM1 gene product will reconstitute an activeGAL4 protein and thereby drive expression of the HIS3 gene. Coloniesthat express HIS3 can be detected by their growth on petri dishescontaining semi-solid agar based media lacking histidine. The cDNA canthen be purified from these strains, and used to produce and isolate thebait PCM1 gene-interacting protein using techniques routinely practicedin the art.

(3) Assays for Compounds that Interfere with PCM1 Gene ProductMacromolecule Interaction

Assays for identifying compounds which interfere with PCM1 gene productmacromolecule are described below.

PCM1 gene products of the invention may, in vivo, interact with one ormore macromolecules, including intracellular macromolecules, such asproteins. Such macromolecules may include, but are not limited to,nucleic acid molecules and those proteins identified via methods such asthose described, above. For purposes of this discussion, themacromolecules are referred to herein as “binding partners”. Compoundsthat disrupt PCM1 binding in this way may be useful in regulating theactivity of the PCM1 gene product, especially mutant PCM1 gene products.Such compounds may include, but are not limited to molecules such aspeptides, and the like, which would be capable of gaining access to aPCM1 gene product.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the PCM1 gene product and itsbinding partner or partners involves preparing a reaction mixturecontaining the PCM1 gene product, and the binding partner underconditions and for a time sufficient to allow the two to interact andbind, thus forming a complex.

In order to test a compound for inhibitory activity, the reactionmixture may be prepared in the presence and absence of the testcompound. The test compound may be initially included in the reactionmixture, or may be added at a time subsequent to the addition of PCM1gene product and its binding partner. Control reaction mixtures areincubated without the test compound or with a placebo. The formation ofany complexes between the PCM1 gene protein and the binding partner isthen detected. The formation of a complex in the control reaction, butnot in the reaction mixture containing the test compound, indicates thatthe compound interferes with the interaction of the PCM1 gene proteinand the interactive binding partner.

Complex formation within reaction mixtures containing the test compoundand normal PCM1 gene protein may also be compared to complex formationwithin reaction mixtures containing the test compound and a mutant PCM1gene protein. This comparison may be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal PCM1 gene proteins.

The assay for compounds that interfere with the interaction of the PCM1gene products and binding partners can be conducted in a heterogeneousor homogeneous format. Heterogeneous assays involve anchoring either thePCM1 gene product or the binding partner onto a solid phase anddetecting complexes anchored on the solid phase at the end of thereaction. In homogeneous assays, the entire reaction is carried out in aliquid phase. In either approach, the order of addition of reactants canbe varied to obtain different information about the compounds beingtested. For example, test compounds that interfere with the interactionbetween the PCM1 gene products and the binding partners, e.g., bycompetition, can be identified by conducting the reaction in thepresence of the test substance; i.e., by adding the test substance tothe reaction mixture prior to or simultaneously with the PCM1 geneprotein and interactive intracellular binding partner. Alternatively,test compounds that disrupt preformed complexes, e.g., compounds withhigher binding constants that displace one of the components from thecomplex, can be tested by adding the test compound to the reactionmixture after complexes have been formed. The various formats aredescribed briefly below.

In a heterogeneous assay system, either the PCM1 gene product or theinteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtitre plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the PCM1 gene product or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored may be used to anchor the species to the solid surface.The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds that inhibit complex formation or that disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the PCM1 gene protein andthe interactive binding partner is prepared in which either the PCM1gene product or its binding partners is labeled, but the signalgenerated by the label is quenched due to complex formation (see, e.g.,U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt PCM1 gene protein/binding partner interactioncan be identified.

In a particular embodiment, the PCM1 gene product can be prepared forimmobilization using recombinant DNA techniques described herein. Forexample, the PCM1 coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive binding partner can bepurified and used to raise a monoclonal antibody, using methodsroutinely practiced in the art, e.g., such as described herein.

This antibody can be labeled with the radioactive isotope ¹²⁵I, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-PCM1 fusion protein can be anchored toglutathione-agarose beads. The interactive binding partner can then beadded in the presence or absence of the test compound in a manner thatallows interaction and binding to occur. At the end of the reactionperiod, unbound material can be washed away, and the labeled monoclonalantibody can be added to the system and allowed to bind to the complexedcomponents. The interaction between the PCM1 gene protein and theinteractive binding partner can be detected by measuring the amount ofradioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-PCM1 gene fusion protein and the interactivebinding partner can be mixed together in liquid in the absence of thesolid glutathione-agarose beads. The test compound can be added eitherduring or after the species are allowed to interact. This mixture canthen be added to the glutathione-agarose beads and unbound material iswashed away. Again the extent of inhibition of the PCM1 geneproduct/binding partner interaction can be detected by adding thelabeled antibody and measuring the radioactivity associated with thebeads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the PCM1 protein and/or the interactive or binding partner (in caseswhere the binding partner is a protein), in place of one or both of thefull length proteins. Any number of methods routinely practiced in theart can be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labelled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the segmentscan be engineered to express peptide fragments of the protein, which canthen be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a PCM1 gene product can beanchored to a solid material as described herein by making a GST-PCM1fusion protein and allowing it to bind to glutathione agarose beads. Theinteractive binding partner obtained can be labeled with a radioactiveisotope, such as ³⁵S, and cleaved with a proteolytic enzyme such astrypsin. Cleavage products can then be added to the anchored GST-PCM1fusion protein and allowed to bind. After washing away unbound peptides,labelled bound material, representing the binding partner bindingdomain, can be eluted, purified, and analyzed for amino acid sequence bywell-known methods. Peptides so identified can be produced syntheticallyor fused to appropriate facilitative proteins using recombinant DNAtechnology.

(4) Methods for Identification of Compounds Which Modulate the Level ofExpression of the PCM1 Gene

Broadly, the level of expression of the PCM1 gene may be determined bymeasuring the amount of PCM1 mRNA, or polypeptide product. Suitabletechniques are described in the section on “Diagnostic methods”.

Alternatively, compounds which modulate the level of PCM1 geneexpression may be identified using a reporter gene system. In such asystem, the PCM1 nucleic acid sequence, or portion thereof (such as thepromoter) is operably linked to a reporter gene.

Use of a reporter gene facilitates determination of the level of geneexpression by reference to protein production. The reporter genepreferably encodes an enzyme which catalyses a reaction which produces adetectable signal, preferably a visually detectable signal, such as acoloured product. Many examples are known, including β-galactosidase andluciferase. β-galactosidase activity may be assayed by production ofblue colour on substrate, the assay being by eye or by use of aspectro-photometer to measure absorbance. Fluorescence, for example thatproduced as a result of luciferase activity, may be quantitated using aspectrophotometer. Radioactive assays may be used, for instance usingchloramphenicol acetyltransferase, which may also be used innon-radioactive assays. The presence and/or amount of gene productresulting from expression from the reporter gene may be determined usinga molecule able to bind the product, such as an antibody or fragmentthereof. The binding molecule may be labelled directly or indirectlyusing any standard technique.

Those skilled in the art are well aware of a multitude of possiblereporter genes and assay techniques which may be used to determinepromoter activity. Any suitable reporter/assay may be used and it shouldbe appreciated that no particular choice is essential to or a limitationof the present invention.

(5) Assays for Identification of Compounds that Ameliorate a PCM1Disorder or a Neuropsychiatric Disorder

Compounds, including but not limited to binding compounds identified viaassay techniques such as those described, above, may be tested for theability to ameliorate symptoms of schizophrenia or disorder of thoughtand/or mood, including thought disorder, and neuropsychiatric disordersincluding delusional disorders, paraphrenia, paranoid psychosis,schizotypal disorder, schizoaffective disorder, schizoaffectiveschizophrenia, psychogenic psychosis, catatonia, periodic schizophrenia,cycloid psychosis, schizoid personality disorder, paranoid personalitydisorder, schizophrenia related affective disorders and subtypes ofunipolar affective disorder.

It should be noted that the assays described herein can identifycompounds that affect PCM1 gene activity by either affecting PCM1 geneexpression or by affecting the level of PCM1 gene product activity. Forexample, compounds may be identified that are involved in another stepin the pathway in which the PCM1 gene and/or PCM1 gene product isinvolved and, by affecting this same pathway may modulate the effect ofPCM1 on the development of a neuropsychiatric disorder such asschizophrenia. Such compounds can be used as part of a therapeuticmethod for the treatment of the disorder.

First, cell-based systems can be used to identify compounds that may actto ameliorate symptoms of a PCM1 disorder or a neuropsychiatricdisorder, such as schizophrenia. Such cell systems can include, forexample, recombinant or non-recombinant cell, such as cell lines, thatexpress the PCM1 gene.

In utilizing such cell systems, cells that express PCM1 may be exposedto a compound suspected of exhibiting an ability to ameliorate symptomsof a PCM1 disorder or a neuropsychiatric disorder, such asschizophrenia, at a sufficient concentration and for a sufficient timeto elicit such an amelioration of such symptoms in the exposed cells.After exposure, the cells can be assayed to measure alterations in theexpression of the PCM1 gene, e.g., by assaying cell lysates for PCM1mRNA transcripts (e.g., by Northern analysis) or for PCM1 gene productsexpressed by the cell; compounds that modulate expression of the PCM1gene are good candidates as therapeutics. Alternatively, the cells areexamined to determine whether one or more cellular phenotypes associatedwith an PCM1 disorder or a neuropsychiatric disorder, such asschizophrenia, has been altered to resemble a more normal or unimpaired,unaffected phenotype, or a phenotype more likely to produce a lowerincidence or severity of disorder symptoms.

In addition, animal-based systems or models for a PCM1 disorder or aneuropsychiatric disorder, such as schizophrenia, which may include, forexample, PCM1 mice, may be used to identify compounds capable ofameliorating symptoms of the disorder. Such animal models may be used astest substrates for the identification of drugs, pharmaceuticals,therapies and interventions that may be effective in treating suchdisorders. For example, animal models may be exposed to a compoundsuspected of exhibiting an ability to ameliorate symptoms, at asufficient concentration and for a sufficient time to elicit such anamelioration of symptoms of a PCM1 disorder or a neuropsychiatricdisorder, such as schizophrenia, in the exposed animals. The response ofthe animals to the exposure may be monitored by assessing the reversalof such symptoms.

With regard to intervention, any treatments that reverse any aspect ofsymptoms of a PCM1 disorder or a neuropsychiatric disorder, such asschizophrenia, should be considered as candidates for human therapeuticintervention in such a disorder. Dosages of test agents may bedetermined by deriving dose-response curves, as discussed below.

Methods of Treatment of Schizophrenia and Compounds for Use in SuchMethods

Methods of treatment of schizophrenia may comprise administeringcompounds which modulate the expression of a mammalian PCM1 gene and/ormodulate the synthesis or activity of a mammalian PCM1 gene product. Inthis way the administration of such compounds results in theamelioration of the symptoms of the disorder.

Such methods may comprise supplying the mammal with a nucleic acidmolecule encoding an unimpaired PCM1 gene product such that anunimpaired PCM1 gene product is expressed and symptoms of the disorderare ameliorated. Such methods maybe useful where the disorder is causedby mutations in the PCM1 gene.

Alternatively, or additionally such methods may comprise supplying themammal with a cell comprising a nucleic acid molecule that encodes anunimpaired PCM1 gene product such that the cell expresses the unimpairedPCM1 gene product and symptoms of the disorder are ameliorated.

In cases in which a loss of normal PCM1 gene product function results inthe development of a schizophrenia, an increase in PCM1 gene productactivity would facilitate progress towards an asymptomatic state inindividuals exhibiting a deficient level of PCM1 gene expression and/orPCM1 gene product activity. Methods for enhancing the expression orsynthesis of PCM1 are described below.

Symptoms of schizophrenia, may be ameliorated by administering acompound that modulates the level of PCM1 gene expression and/or PCM1gene product activity. Such compounds for use in the treatment ofschizophrenia represent further aspects of the invention.

Suitable compounds for use in such methods may include compounds, inparticular drugs, reported to ameliorate or exacerbate the symptoms of aneuropsychiatric disorder, such as schizophrenia. Such compounds includeantidepressants such as lithium salts, flupenthixol, risperidone,clozapine, quetiapine, olanzapine, haloperidol, droperidol,chlorpromazine, prochloperazine, phenothiazaine derivatives, promazine,trifluopromazine, butyrophenone derivatives, thioxanthine derivativessuch as chlorprothixene and dibenzodiazepines and antipsychoticantiepileptic drugs such carbamazepine, and valproic acid, reserpine.Psychotogenic drugs such as LSD, bromocriptine, apomorphine,amphetamine, methylphenidate, methylamphetaime, ketamine, Many of thesedrugs are used in combination.

(1) Inhibitory Antisense, Ribozyme and Triple Helix Approaches

In another embodiment, symptoms of schizophrenia may be ameliorated bydecreasing the level of PCM1 gene expression and/or PCM1 gene productactivity by using PCM1 gene sequences in conjunction with well-knownantisense, gene “knock-out,” ribozyme and/or triple helix methods todecrease the level of PCM1 gene expression. Among the compounds that mayexhibit the ability to modulate the activity, expression or synthesis ofthe PCM1 gene, including the ability to ameliorate the symptoms of aPCM1 disorder or a neuropsychiatric disorder, such as schizophrenia, areantisense, ribozyme, and triple helix molecules. Such molecules may bedesigned to reduce or inhibit either unimpaired, or if appropriate,mutant target gene activity. Techniques for the production and use ofsuch molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense approaches involve the design of oligonucleotides that arecomplementary to a target gene mRNA. The antisense oligonucleotides willbind to the complementary target gene mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired.

A sequence “complementary” to a portion of an RNA, as referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids, a single strand of the duplexDNA may thus be tested, or triplex formation may be assayed. The abilityto hybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

In one embodiment, oligonucleotides complementary to non-coding regionsof the PCM1 gene could be used in an antisense approach to inhibittranslation of endogenous PCM1 mRNA. Antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86,6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci. 84,648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents (see,e.g., Krol et al., 1988, BioTechniques 6, 958-976) or intercalatingagents (see, e.g., Zon, 1988, Pharm. Res. 5, 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methylguanine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier, et al.,1987, Nucl. Acids Res. 15, 6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res. 15,6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al., 1987, FEBSLett. 215, 327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein, et al. (1988, Nucl. Acids Res. 16, 3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85, 7448-7451), etc.

While antisense nucleotides complementary to the target gene codingregion sequence could be used, those complementary to the transcribed,untranslated region are most preferred. For example, antisenseoligonucleotides having the following sequences can be utilized inaccordance with the invention:

Antisense molecules should be delivered to cells that express the targetgene in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous target gene transcripts andthereby prevent translation of the target gene mRNA. For example, avector can be introduced e.g., such that it is taken up by a cell anddirects the transcription of an antisense RNA. Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in mammalian cells. Expression of thesequence encoding the antisense RNA can be by any promoter known in theart to act in mammalian, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include but are not limitedto: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290, 304-310), the promoter contained in the 31 long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22, 787-797), theherpes thymidine kinase promoter (Wagner, et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of themetallothionein gene (Brinster, et al., 1982, Nature 296, 39-42), etc.Any type of plasmid, cosmid, YAC or viral vector can be used to preparethe recombinant DNA construct which can be introduced directly into thetissue site. Alternatively, viral vectors can be used that selectivelyinfect the desired tissue, in which case administration may beaccomplished by another route (e.g., systemically).

Ribozyme molecules designed to catalytically cleave target gene mRNAtranscripts can also be used to prevent translation of target gene mRNAand, therefore, expression of target gene product. (See, e.g., PCTInternational Publication WO90/11364, published Oct. 4, 1990; Sarver, etal., 1990, Science 247, 1222-1225).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. (For a review, see Rossi, 1994, Current Biology 4,469-471). The mechanism of ribozyme action involves sequence specifichybridization of the ribozyme molecule to complementary target RNA,followed by an endonucleolytic cleavage event. The composition ofribozyme molecules must include one or more sequences complementary tothe target gene mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see, e.g., U.S. Pat.No. 5,093,246, which is incorporated herein by reference in itsentirety.

While ribozymes that cleave mRNA at site specific recognition sequencescan be used to destroy target gene mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Myers, 1995, Molecular Biology andBiotechnology: A Comprehensive Desk Reference, VCH Publishers, New York,(see especially FIG. 4, page 833) and in Haseloff and Gerlach, 1988,Nature, 334, 585-591, which is incorporated herein by reference in itsentirety.

Preferably the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the target gene mRNA, i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts. For example, hammerhead ribozymes maybe used.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onethat occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and that has been extensively described by Thomas Cech andcollaborators (Zaug, et al., 1984, Science, 224, 574-578; Zaug and Cech,1986, Science, 231, 470-475; Zaug, et al., 1986, Nature, 324, 429-433;published International patent application No. WO 88/04300 by UniversityPatents Inc.; Been and Cech, 1986, Cell, 47, 207-216). The Cech-typeribozymes have an eight base pair active site which hybridizes to atarget RNA sequence whereafter cleavage of the target RNA takes place.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells that express the target gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous target gene messagesand inhibit translation. Because ribozymes unlike antisense molecules,are catalytic, a lower intracellular concentration is required forefficiency.

Endogenous target gene expression can also be reduced by inactivating or“knocking out” the target gene or its promoter using targeted homologousrecombination (e.g., see Smithies, et al., 1985, Nature 317, 230-234;Thomas and Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989,Cell 5, 313-321; each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional target gene (or acompletely unrelated DNA sequence) flanked by DNA homologous to theendogenous target gene (either the coding regions or regulatory regionsof the target gene) can be used, with or without a selectable markerand/or a negative selectable marker, to transfect cells that express thetarget gene in vivo. Insertion of the DNA construct, via targetedhomologous recombination, results in inactivation of the target gene.Such approaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive target gene (e.g., see Thomas andCapecchi, 1987 and Thompson, 1989, supra). However this approach can beadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors.

Alternatively, endogenous target gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the target gene (i.e., the target gene promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the target gene in target cells in the body. (See generally, Helene,1991, Anticancer Drug Des., 6(6), 569-584; Helene, et al., 1992, Ann.N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays 14(12),807-815).

Nucleic acid molecules to be used in triplex helix formation for theinhibition of transcription should be single stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides must bedesigned to promote triple helix formation via Hoogsteen base pairingrules, which generally require sizeable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGC⁺triplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, contain a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

In instances wherein the antisense, ribozyme, and/or triple helixmolecules described herein are utilized to inhibit mutant geneexpression, it is possible that the technique may so efficiently reduceor inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene allelesthat the possibility may arise wherein the concentration of normaltarget gene product present may be lower than is necessary for a normalphenotype. In such cases, to ensure that substantially normal levels oftarget gene activity are maintained, therefore, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity may, be introduced into cells via gene therapymethods such as those described, below, that do not contain sequencessusceptible to whatever antisense, ribozyme, or triple helix treatmentsare being utilized. Alternatively, in instances whereby the target geneencodes an extracellular protein, it may be preferable to co-administernormal target gene protein in order to maintain the requisite level oftarget gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules, as discussed above. These includetechniques for chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

(2) Gene Replacement Therapy

With respect to an increase in the level of normal PCM1 gene expressionand/or PCM1 gene product activity, PCM1 gene nucleic acid sequences,describe above, can, for example, be utilized for the treatment of aPCM1 disorder or a neuropsychiatric disorder, such as schizophrenia.Such treatment can be administered, for example, in the form of genereplacement therapy. Specifically, one or more copies of a normal PCM1gene or a portion of the PCM1 gene that directs the production of a PCM1gene product exhibiting normal PCM1 gene function, may be inserted intothe appropriate cells within a patient, using vectors that include, butare not limited to adenovirus, adeno-associated virus, and retrovirusvectors, in addition to other particles that introduce DNA into cells,such as liposomes.

Because the PCM1 gene is expressed in the brain, such gene replacementtherapy techniques should be capable delivering PCM1 gene sequences tothese cell types within patients. Thus, in one embodiment, techniquesthat are well known to those of skill in the art (see, e.g., PCTPublication No. WO89/10134, published Apr. 25, 1988) can be used toenable PCM1 gene sequences to cross the blood-brain barrier readily andto deliver the sequences to cells in the brain. With respect to deliverythat is capable of crossing the blood-brain barrier, viral vectors suchas, for example, those described above, are preferable. Also includedare methods using liposomes either in vivo ex vivo or in vitro. WhereinPCM1 sense or antisense DNA is delivered to the cytoplasm and nucleus oftarget cells. Liposomes can deliver PCM1 sense or nonsense RNA to humansand the human brain or in mammals through intrathecal delivery either aspart of a viral vector or as DNA conjugated with nuclear localizingproteins or other proteins that increase take up into the cell nucleus.

In another embodiment, techniques for delivery involve directadministration of such PCM1 gene sequences to the site of the cells inwhich the PCM1 gene sequences are to be expressed. Additional methodsthat may be utilized to increase the overall level of PCM1 geneexpression and/or PCM1 gene product activity include the introduction ofappropriate PCM1-expressing cells, preferably autologous cells, into apatient at positions and in numbers that are sufficient to amelioratethe symptoms of a PCM1 disorder or a neuropsychiatric disorder, such asschizophrenia. Such cells may be either recombinant or non-recombinant.

Among the cells that can be administered to increase the overall levelof PCM1 gene expression in a patient are normal cells, preferably braincells and also choroid plexus cells within the CNS which are accessiblethrough intrathecal injections. Alternatively, cells, preferablyautologous cells, can be engineered to express PCM1 gene sequences, andmay then be introduced into a patient in positions appropriate for theamelioration of the symptoms of a PCM1 disorder or a neuropsychiatricdisorder, such as schizophrenia. Alternately, cells that express anunimpaired PCM1 gene and that are from a MHC matched individual can beutilized, and may include, for example, brain cells. The expression ofthe PCM1 gene sequences is controlled by the appropriate gene regulatorysequences to allow such expression in the necessary cell types. Suchgene regulatory sequences are well known to the skilled artisan. Suchcell-based gene therapy techniques are well known to those skilled inthe art, see, e.g., Anderson, U.S. Pat. No. 5,399,349.

When the cells to be administered are non-autologous cells, they can beadministered using well known techniques that prevent a host immuneresponse against the introduced cells from developing. For example, thecells may be introduced in an encapsulated form which, while allowingfor an exchange of components with the immediate extracellularenvironment, does not allow the introduced cells to be recognized by thehost immune system.

Additionally, compounds, such as those identified via techniques such asthose described above, that are capable of modulating PCM1 gene productactivity can be administered using standard techniques that are wellknown to those of skill in the art. In instances in which the compoundsto be administered are to involve an interaction with brain cells, theadministration techniques should include well known ones that allow fora crossing of the blood-brain barrier such as intrathecal injection andconjugation with compounds that allow transfer across the blood brainbarrier.

Pharmaceutical Preparations and Methods of Administration

Compounds that are determined to affect PCM1 gene expression or geneproduct activity can be administered to a patient at therapeuticallyeffective doses to treat or ameliorate schizophrenia.

A therapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of such a disorder.

(1) Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

(2) Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or intrathecal,oral, buccal, parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner. For administration byinhalation, the compounds for use according to the present invention areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Nucleic acid Molecules

Nucleic acid molecules for use in the various aspects of the inventionmay be provided isolated and/or purified from their natural environment,in substantially pure or homogeneous form, or free or substantially freeof other nucleic acids of the species of origin. Where used herein, theterm “isolated” encompasses all of these possibilities. The nucleic acidmolecules may be wholly or partially synthetic. In particular they maybe recombinant in that nucleic acid sequences which are not foundtogether in nature (do not run contiguously) have been ligated orotherwise combined artificially. Alternatively they may have beensynthesised directly e.g. using an automated synthesiser. Nucleic acidaccording to the present invention may include cDNA, RNA and modifiednucleic acids or nucleic acid analogs. Where a DNA sequence isspecified, e.g. with reference to a figure, unless context requiresotherwise the RNA equivalent, with U substituted for T where it occurs,is encompassed. Where a nucleic acid (or nucleotide sequence) of theinvention is referred to herein, the complement of that nucleic acid (ornucleotide sequence) will also be embraced by the invention. The‘complement’ in each case is the same length as the reference, but is100% complementary thereto whereby by each nucleotide is capable of basepairing with its counterpart i.e. G to C, and A to T or U.

The nucleic acid may be in the form of a recombinant, and preferablyreplicable vector. Preferably the nucleic acid is under the control of,and operably linked to regulatory elements which are capable ofdirecting expression from the PCM1 nucleic acid sequence.

As used herein, regulatory elements include but are not limited to,inducible and non-inducible promoters, enhancers, operators and otherelements known to those skilled in the art that drive and regulateexpression. Such regulatory elements include but are not limited to thecytomegalovirus hCMV immediate early gene, the early or late promotersof SV40 adenovirus,the lac system, the trp system, the TAC system, theTRC system, the CRE/LOX system, the major operator and promoter regionsof phage A, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, and thepromoters, of the yeast α-mating factors.

Polypeptides and Proteins

Polypeptides for use according to the various aspects of the inventionmay be prepared by chemical synthesis, or by using recombinanttechnology, as is understood by the person skilled in the art.

A variety of host-expression vector systems may be utilized to expressthe PCM1 gene coding sequences. Such host-expression systems representvehicles by which the coding sequences of interest may be produced andsubsequently purified, but also represent cells that may, whentransformed or transfected with the appropriate nucleotide codingsequences, exhibit the PCM1 gene product of the invention in situ.Suitable host-expression systems include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus); plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the PCM1gene product being expressed. For example, when a large quantity of sucha protein is to be produced, e.g., for the generation of pharmaceuticalcompositions of PCM1 protein or for raising antibodies to the PCM1protein, vectors that direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2, 1791), in which the PCM1 geneproduct coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13, 3101-3109;Van Heeke and Schuster, 1989, J. Biol. Chem. 264, 5503-5509); and thelike. PGEX vectors may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica, nuclear polyhedrosis virus(AcNPV) may be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The PCM1 gene coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an ACNPVpromoter (for example the polyhedrin promoter). Successful insertion ofPCM1 gene coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed. (e.g., see Smith, et al., 1983,J. Virol. 46, 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the PCM1 gene coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing PCM1 gene product in infected hosts. (e.g., See Logan andShenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-3659).

Specific initiation signals may also be required for efficienttranslation of inserted PCM1 gene product coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire PCM1 gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. However, incases where only a portion of the PCM1 gene coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, should be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner, et al., 1987,Methods in Enzymol. 153, 516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express thePCM1 gene product may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express the PCM1 geneproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the PCM1 gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11, 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, 1962, Proc. Natl. Acad. Sci. USA 48, 2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22, 817) genes canbe employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77, 3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78, 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78, 2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150, 1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30, 147).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht, et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88,8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

The PCM1 gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate PCM1 transgenic animals. The term “transgenic,” as used herein,refers to animals expressing PCM1 gene sequences from a differentspecies (e.g., mice expressing human PCM1 sequences), as well as animalsthat have been genetically engineered to overexpress endogenous (i.e.,same species) PCM1 sequences or animals that have been geneticallyengineered to no longer express endogenous PCM1 gene sequences (i.e.,“knock-out” animals), and their progeny.

Any technique known in the art may be used to introduce an PCM1 genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten, etal., 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); gene targeting inembryonic stem cells (Thompson, et al., 1989, Cell 56, 313-321);electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3, 1803-1814);and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57,717-723) (For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115; 171-229)

Once transgenic animals have been generated, the expression of therecombinant PCM1 gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include but are not limited to Northern blot analysis oftissue samples obtained from the animal, in situ hybridization analysis,and RT-PCR (reverse transcriptase PCR). Samples of PCM1 gene-expressingtissue, may also be evaluated immunocytochemically using antibodiesspecific for the PCM1 transgene product.

Antibodies

Antibodies capable of specifically recognising the polypeptide encodedby the PCM1 gene, or capable of specifically recognising fragments orepitopes of the polypeptide encoded by the PCM1 gene, may be used in thevarious aspects of the invention.

For example, such antibodies may be used in the detection of a PCM1 geneproduct in an biological sample and may, therefore, be utilized as partof a diagnostic or prognostic technique whereby patients may be testedfor abnormal levels of PCM1 gene products, and/or for the presence ofabnormal forms of such gene products.

Such antibodies may also be utilized in conjunction with, for example,compound screening schemes, as described herein, for the evaluation ofthe effect of test compounds on PCM1 gene product levels and/oractivity.

Such antibodies may be used to inhibit abnormal PCM1 gene productactivity. Thus, such antibodies may, therefore, be used in a method oftreatment or therapy for schizophrenia.

For example, antibodies may be used in a gene therapy technique,described herein, e.g., to evaluate the normal and/or engineeredPCM1-expressing cells prior to their introduction into the patient.

Such antibodies may include, but are not limited to, polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above.

For the production of antibodies against a PCM1 gene product, varioushost animals may be immunized by injection with a PCM1 gene product, ora portion thereof. Such host animals may include, but are not limited torabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and otentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a PCM1 gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with PCM1 geneproduct supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256, 495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4, 72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80, 2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., 1984, Proc. Natl. Acad. Sci., 81,6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al.,1985, Nature, 314, 452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimaeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss etal., U.S. Pat. No. 4,816,397)

In addition, techniques have been developed for the production ofhumanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089, whichis incorporated herein by reference in its entirety.) An immunoglobuinlight or heavy chain variable region consists of a “framework” regioninterrupted by three hypervariable regions, referred to ascomplementarity determining regions (CDRs). The extent of the frameworkregion and CDRs have been precisely defined (see, “Sequences of Proteinsof Immunological Interest”, Kabat, E. et al., U.S. Department of Healthand Human Services (1983). Briefly, humanized antibodies are antibodymolecules from non-human species having one or more CDRs from thenon-human species and a framework region from a human immunoglobulinmolecule.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242, 423-426;Huston, et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879-5883; andWard, et al., 1989, Nature 334, 544-546) can be adapted to producesingle chain antibodies against PCM1 gene products. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule and the Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse, etal., 1989, Science, 246, 1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

Variants and Derivatives

Where the invention relates to methods which use the PCM1 gene, or touse of the PCM1 gene, these methods (and uses) may use a variant of thePCM 1 gene. Such variants are now discussed in more detail.

A variant nucleic acid molecule shares homology with, or is identicalto, all or part of the coding sequence discussed above. Generally,variants may encode, or be used to isolate or amplify nucleic acidswhich encode, polypeptides which are have the same activity as thepolypeptide encoded by the PCM 1 nucleic acid sequence and/or which willspecifically bind to an antibody raised against the polypeptide encodedby the PCM 1 nucleic acid sequence.

Variants of the present invention can be artificial nucleic acids (i.e.containing sequences which have not originated naturally) which can beprepared by the skilled person in the light of the present disclosure.Alternatively they may be novel, naturally occurring, nucleic acids,which may be isolatable using the sequences disclosed herein.

Thus a variant may be a distinctive part or fragment (however produced)corresponding to a portion of the PCM1 gene sequence. The fragments mayencode particular functional parts of the polypeptide. Equally thefragments may have utility in probing for, or amplifying, the sequenceprovided or closely related ones.

Also included are nucleic acids which have been extended at the 3′ or 5′terminus.

Artificial variants (derivatives) may be prepared by those skilled inthe art, for instance by site directed or random mutagenesis, or bydirect synthesis. The term ‘variant’ nucleic acid as used hereinencompasses all of these possibilities.

Some of the aspects of the present invention relating to variants willnow be discussed in more detail.

Homology (i.e. similarity or identity) may be as defined using sequencecomparisons are made using FASTA and FASTP (see Pearson & Lipman, 1988.Methods in Enzymology 183: 63-98). Parameters are preferably set, usingthe default matrix, as follows: Gapopen (penalty for the first residuein a gap): −12 for proteins/−16 for DNA; Gapext (penalty for additionalresidues in a gap): −2 for proteins/−4 ′ for DNA; KTUP word length: 2for proteins/6 for DNA. Homology may be at the nucleotide sequenceand/or encoded amino acid sequence level. Preferably, the nucleic acidand/or amino acid sequence shares at least about 60%, or 70%, or 80%homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99%homology with the sequence of the PCM1 gene.

Thus a variant polypeptide in accordance with the present invention mayinclude within its sequence, a single amino acid or 2, 3, 4, 5, 6, 7, 8,or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater thanabout 50, 60, 70, 80, 90, 100, 200 changes, compared with thepolypeptide encoded by the PCM1 gene. In addition to one or more changeswithin the amino acid sequence shown, a variant polypeptide may includeadditional amino acids at the C-terminus and/or N-terminus. Naturally,regarding nucleic acid variants, changes to the nucleic acid which makeno difference to the encoded polypeptide (i.e. ‘degenerativelyequivalent’) are included within the scope of the present invention.

Changes to a sequence, to produce a derivative, may be by one or more ofaddition, insertion, deletion or substitution of one or more nucleotidesin the nucleic acid, leading to the addition, insertion, deletion orsubstitution of one or more amino acids in the encoded polypeptide.

Changes may be desirable for a number of reasons, including introducingor removing the following features: restriction endonuclease sequences;codon usage; other sites which are required for post translationmodification; cleavage sites in the encoded polypeptide; motifs in theencoded polypeptide(e.g. binding sites) Leader or other targetingsequences (e.g. hydrophobic anchoring regions) may be added or removedfrom the expressed protein to determine its location followingexpression. All of these may assist in efficiently cloning andexpressing an active polypeptide in recombinant form (as describedbelow).

Other desirable mutations may be random or site directed mutagenesis inorder to alter the activity (e.g. specificity) or stability of theencoded polypeptide.

Changes may be by way of conservative variation, i.e. substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine. As is well known to those skilled in the art,altering the primary structure of a polypeptide by a conservativesubstitution may not significantly alter the activity of that peptidebecause the side-chain of the amino acid which is inserted into thesequence may be able to form similar bonds and contacts as the sidechain of the amino acid which has been substituted out. This is so evenwhen the substitution is in a region which is critical in determiningthe peptides conformation.

Also included are variants having non-conservative substitutions. As iswell known to those skilled in the art, substitutions to regions of apeptide which are not critical in determining its conformation may notgreatly affect its activity because they do not greatly alter thepeptide's three dimensional structure.

In regions which are critical in determining the peptides conformationor activity such changes may confer advantageous properties on thepolypeptide. Indeed, changes such as those described above may conferslightly advantageous properties on the peptide e.g. altered stabilityor specificity.

The invention will now be described with reference to the followingnon-limiting examples and Figures.

FIGURES, TABLES

FIG. 1 shows the longest open reading frame of Homo sapienspericentriolar material 1 (PCM1) mRNA and the translated product. Thecoding sequence has a length of 6075 nt and the translated product is2024 aa long. The translation initiation codon (atg) is at position 410while the stop codon (tga) is at position 6482 of the PCM1 mRNA.

FIG. 2 shows the LI-COR AlignIR alignment report for a novel DNA variantidentified in intronic sequence 3′ to exon 4 of PCM1 (position 80254 ofthe AB020866 clone). The sequence of clone AB020866 was the referencegenomic sequence obtained from the Entrez nucleotide database and can beseen at the first line (ex4gnm). The variation occurs at nucleotideposition 80254 of the AB020866 clone (marked X). The consensus sequenceobtained from AlignIR can be seen at the last line (Consensus).Heterozygotes are marked R, G homozygotes are marked G, A homozygotesare marked A.

FIG. 3 shows the LI-COR AlignIR alignement report for a novel DNAvariant identified in exon 4 of PCM1 (position 80123 of the AB020866clone). The sequence of the cDNA of PCM1 (NM_(—)006197) can be seen atthe first line (ex4cDNA) while the clone AB020866 was the referencegenomic sequence and can be seen at the second line (ex4gnm). Bothsequences were obtained from the Entrez nucleotide database. Thevariation occurs at nucleotide position 1100 of the cDNA and 80123 ofthe AB020866 clone and is marked X. The consensus sequence obtained fromAlignIR can be seen at the last line (Consensus). Heterozygotes, areshown as R, G homozygotes as G.

FIG. 4 shows the LI-COR AlignIR alignment report for a novel DNA variantidentified in intronic sequence 5′ to exon 5 of PCM1 (position 87366 ofthe AB020866 clone). The, sequence of clone AB020866 was the referencegenomic sequence obtained from the Entrez nucleotide database and can beseen at the first line (ex5gnm). The variation occurs at nucleotideposition 87366 of the AB020866 clone (marked X). The consensus sequenceobtained from AlignIR can be seen at the last line (Consensus).Heterozygotes are marked as Y, C homozygotes are marked as C, Thomozygotes are marked as T.

FIG. 5 shows the AlignIR alignment report for a novel DNA variantidentified at position 87507 of the AB020866 clone). The sequence ofclone AB020866 was the reference genomic sequence obtained from theEntrez nucleotide database and can be seen at the first line (ex5gnm).The variation occurs at nucleotide position 87507 of the AB020866 cloneis marked with an X. The consensus sequence obtained from AlignIR can beseen at the last line (Consensus). Heterozygotes are marked R, Ghomozygotes are marked G, A homozygotes are marked A.

FIG. 6 (Table 4) shows the exon-intron boundaries referred to in Example2.

DETAILED DESCRIPTION OF INVENTION

The studies are described that, first, define an interval ofapproximately 500 kb on the short arm of human chromosome 8 within whicha region associated with a neuropsychiatric disorder is located and,second, identified a novel mutation within the gene referred to hereinas PCM1-EX41, which lies within this region and which is involved inneuropsychiatric disorders.

EXAMPLE 1 Linkage Disequilibrium

Materials and Methods

Linkage Disequilibrium.

Linkage disequilibrium (LD) studies were performed using DNA from apopulation sample of neuropsychiatric disorder (schizophrenia) patients.The population sample and LD techniques were as described as below. Thepresent LD study took advantage of the discovery of additional physicalmarkers identified via the physical mapping and sequencing techniquesdescribed below.

Bacterial Artificial Chromosome (BAC) Mapping.

For physical mapping, bacterial artificial chromosomes (BACs) containinghuman sequences were mapped to the region being analyzed based onpublicly available maps (Human genome database, Toronto 1999 and ). TheBACs were then ordered and contig reconstructed by performing standardmapping with microsatellite markers and polymorphic SNP's that werediscovered by HMDG and EB and.

Bacterial artificial chromosome (BAC) mapping. Sequences flanking amicrosatellite polymorphism were used to screen a human BAC library. Theends of the BACs were cloned and subclones were sequenced. From one suchBAC, and from raw sequence data additional microsatellites wereidentified. A microsatellite sequence from the sublibrary was identifiedby corresponding microsatellite probes. Sequences around such repeatswere obtained to enable development of PCR primers for genomic DNA.

Fluoresecent in situ hybridization (FISH) was used map the new BAC tothe region being analyzed.

The resulting sequences were then compared to public DNA and proteindatabases using BLAST algorithms (Altschul, et al., 1990, J. Mclec.Biol., 215, 403-410).

Results

Genetic regions involved in schizophrenia had previously been reportedto map to portions of the short (8p) arm of human chromosome 8.Including a broad genetic region of about 60 cM between markers on mostof the short arm of chromosome 8p.

Prior to attempting to identify gene sequences, studies were performedto further narrow the neuropsychiatric disorder region.

Specifically, a linkage disequilibrium (LD) analysis was performed usingpopulation samples and techniques as described above, which tookadvantage of the additional physical markers identified via the physicalmapping and sequencing techniques described below.

In order to provide the precise order of genetic markers necessary forlinkage and LD mapping, and to guide new microsatellite markerdevelopment for finer mapping, a high resolution physical map of the8p21-22.3 candidate region was developed (Table 1). Elevenpolymorphisms, seven of which are described here for the first time (seeTable 2), were genotyped in a sample of 134 schizophrenia patients of UKancestry and 316 ethnically matched normal controls. TABLE 1 The clonesthat constitute the NT 000501 contig (121,0381 bases) of the human8p21.3-p22 sequence are shown. The position of seven novelmicrosatellite markers which developed from this sequence together withtwo already known markers (D8S261 and AFM333th1) are shown as well astheir respective clones and the intermarker distances between the ninemarkers. POSITION INTERMARKER IN THE DISTANCES CLONES SIZE (bp) MARKERSCLONE (bp) (bp) AB020858.1 100,000 AB020859.1 100,000 AB020860.1 100,000AB020861.1 100,000 AB020862.1 100,000 AB020863.1 156,909 D8S2618 14,588178,562 AB020864.1 100,000 D8S2613 36,241 90,549 AB020865.1 100,000D8S2614 26,790 73,402 AB020866.1 100,000 D8S2615 192 86,911 D8S261687,103 21,981 AB020867.1 100,000 D8S261 9,084 6,150 AFM333th1 15,23462,533 D8S2617 77,767 28,400 AB020868.1 153,472 D8S2612 6,167

TABLE 2 Primer sequences and allele set of the novel microsatelliterepeats identified in the NT 000501 contig on chromosome 8p21.3-22D8S2612 Primers: 5′AAT TCC CCA AAC AAA ACA ACA3′ 5′AGG CTA TCC TTT CCTCAG CA3′ Alleles Length (bp) (CA)n Frequency 1 161 18 0.0112 2 159 170.0594 3 157 16 0.1614 4 155 15 0.2578 5 153 14 0.4283 6 151 13 0.0751 7149 12 0.0000 8 147 11 0.0000 9 145 10 0.0067 Heterozygosity: 0.7147 No.of chromosomes: 892 D8S2617 Primers: 5′ATG TTC AGC CAC CAT CGT CT3′5′CAG TGT CGC TGG AAA GTT GA3′ Alleles Length (bp) (CA)n Frequency 1 20325 0.0146 2 201 24 0.0822 3 199 23 0.1216 4 197 22 0.0619 5 195 210.2759 6 193 20 0.4358 7 191 19 0.0068 8 189 18 0.0000 9 187 17 0.000010 185 16 0.0011 Heterozygosity: 0.7083 No. of chromosomes: 888 D8S2616Primers: 5′TCC CGA AGT GCT AGG ATT ACA3′ 5′GCT CAG CAG GAA GAG GAA TG3′Alleles Length (bp) (CA)n Frequency 3 213 24 0.0011 4 211 23 0.0113 5209 22 0.0317 6 207 21 0.0588 7 205 20 0.1301 8 203 19 0.5848 9 201 180.0532 10 199 17 0.1199 11 197 16 0.0011 12 195 15 0.0057 13 193 140.0000 14 191 13 0.0011 15 189 12 0.0011 Heterozygosity: 0.6192 No. ofchromosomes: 884 D8S2615 Primers: 5′AGA GGC CAG GCA CAA AAG TA3′ 5′AACATT CCA GCA TCC CAA AG3′ Alleles Length (bp) (CA)n Frequency 1 205 240.8807 2 203 23 0.0148 3 201 22 0.0875 4 119 21 0.0000 5 117 19 0.0000 6115 18 0.0136 7 113 17 0.0011 8 111 16 0.0000 9 99 15 0.0000 10 97 140.0023 Heterozygosity: 0.2163 No. of chromosomes: 880 D8S2614 Primers:5′GAC CCA CTG CCA CAC TCT TT3′ 5′GGA GTG CGG CAT GAA ATT AT3′ AllelesLength (bp) (CA)n Frequency 1 194 22 0.0057 2 192 21 0.0588 3 190 200.4570 4 188 19 0.0916 5 186 18 0.1437 6 184 17 0.0045 7 182 16 0.2285 8180 15 0.0000 9 178 14 0.0011 10 176 13 0.0079 11 174 12 0.0011Heterozygosity: 0.7063 No. of chromosomes: 884 D8S2613 Primers: 5′ATATGT ATA CAA TGT GTA TCT GTA TC3′ 5′CCT TTT AGT TCC CAT TCC CAT T3′Alleles Length (bp) (CA)n Frequency 1 133 16 0.0275 2 131 15 0.2002 3129 14 0.1167 4 127 13 0.0000 5 125 12 0.6362 6 123 11 0.0000 7 121 100.0046 8 119 9 0.0000 9 117 8 0.0137 10 115 7 0.0011 Heterozygosity:0.5406 No. of chromosomes: 874 D8S2618 Primers: 5′TGA TGC AGG AGA ATTGCT TG3′ 5′CCT ACT TGG CTG GGA TTC TG3′ Alleles Length (bp) (CA)nFrequency 1 182 24 0.00457 2 180 23 0.00686 3 178 22 0.05148 4 176 210.01487 5 174 20 0.07208 6 172 19 0.04462 7 170 18 0.02745 8 168 170.36041 9 166 16 0.02288 10 164 15 0.00228 11 162 14 0.01029 12 160 130.37185 13 158 12 0.00000 14 156 11 0.00000 15 154 10 0.00915 16 152 90.00114 Heterozygosity: 0.72023 No. of chromosomes: 874

Three neighbouring polymorphisms, D8S66181, D8S6687 and D8S261, spanninga region of approximately 108 kb demonstrated significant evidence forallelic association with schizophrenia with a p-value 0.004, 0.024 and0.055 respectively after a Monte Carlo correction for multiple alleles(Table 2). Statistically significant marker-to-marker linkagedisequilibrium was found between the positively associated markers inthis region and overall correlated well with the physical distance ofthe markers. The new microsatellite and SNP markers identified viasequencing and genotyping were genotyped in an LD analysis of samplescollected from cases of schizophrenia. The results of this LD analysisnarrowed down the chromosome 8p arm region within which a gene involvedin neuropsychiatric disorders lies to an interval of 70 kb flanking thepublicly available marker D8S261. The gene PCM1 was the only genemapping exactly at the positions of maximum disequilibrium over the 70kb region thus showing it can be involved in neuropsychiatric disorders.TABLE 3 CLUMP test results for association between schizophrenia andalleles at novel polymorphic loci on chromosome 8p21.3-22 in a UKcase-control sample Marker T1 T2 T3 T4 Locus X² p-value X² p-value X²p-value X² p-value (tel→cen) 8.191 0.215 3.439 0.497 3.271 0.287 3.7950.414 D8S2612 D8S2617 6.714 0.465 4.139 0.510 1.965 0.570 1.965 0.797AFM333th1 5.301 0.509 4.108 0.544 2.061 0.512 3.744 0.426 D8S261 16.670.055 10.60 0.150 9.992 0.010 13.82 0.017 D8S2616 19.92 0.024 12.990.048 5.268 0.118 11.91 0.031 D8S2615 15.17 0.004 10.54 0.009 10.540.005 12.12 0.004 D8S2614 6.560 0.720 4.882 0.445 3.551 0.260 4.1640.474 D8S2613 5.449 0.496 2.728 0.608 0.273 0.956 3.937 0.319 D8S261812.36 0.521 7.996 0.423 0.790 0.947 9.345 0.210Where,T1: a straightforward Pearson's χ² statistic of the ‘raw’ contingencytableT2: the χ² statistic of a table with rare alleles grouped together toprevent small expected cell countsT3: the largest of the χ² statistics of 2 × 2 tables each of whichcompares one allele against the rest grouped togetherT4: the largest of the χ² statistics of all possible 2 × 2 tablescomparing any combination of alleles against the rest

The pericentriolar material 1 gene (PCM 1) has been mapped to chromosome8p21.3-33 [Ohata, (1994)Genomics 24, 404-406)

In addition, it is annotated in the Genome Channel and its sequenceoccupies part of clones AB020866 and AB020867 of the NT_(—)000501contig. Two of the associated polymorphisms, D8S2616 and D8S261 liewithin the intronic sequence of this gene while the third polymorphism,D8S2615 resides approximately 75 kb upstream of the translationinitiation codon of this gene. Balczon et al, [Balczon, 1991, Cell MotilCytoskeleton 20:121-135] identified a ˜220 kD centrosome autoantigen. AcDNA encoding the entire protein was isolated from a human fetal livercDNA library. Analysis of the cloned sequence identified an open readingframe of 6,072 nucleotides encoding 2,024 amino acids. From the deducedamino acid sequence, the exact molecular mass of PCM1 was calculated tobe 228,705 daltons [Balczon, 1994]. PCM1 nucleotide and amino acidsequences are shown in FIG. 1.

EXAMPLE 2 Genomic Organization of PCM1

The predicted coding sequence has a translation initiation codon (ATG)located at position 410 of PCM1 mRNA, which is preceded by a Kozakconsensus (CCAXXATGG) initiation sequence [Kozak, 1984, Nature 308:241-246] and a stop codon (TGA) is located at position 6482 of the PCM1mRNA (FIG. 1).

In order to define the exon-intron genomic organization, “BLAST 2sequences” program from the NCBI was used. This is a tool, whichproduces an alignment of two given sequences using the BLAST program. Inthis case, the two sequences were the mRNA sequence of PCM1 (AccessionNo. NM_(—)006197) against the corresponding genomic sequence (clonesAB020866 and AB020867 of the NT_(—)000501 contig) The exon-intronjunctions of the PCM1 transcript derived from these data is shown inTable 4 (FIG. 6). The predicted exon-intron boundaries are in excellentagreement with the “GT-AG rule” that is nucleotides at the exon-intronboundaries are not random but introns always seem to begin with GT andend with AG. In addition, the predicted exons are in agreement with theexons generated by the GRAILEXP program used for gene prediction by theGenome Channel, which is a site that provides automated annotation ofgenomic sequence.

The coding sequence has a length of 6075 nt and the translated productis 2024 aa long. The translation initiation codon (atg) is at position410 while the stop codon (tga) is at position 6482 of the PCM1 mRNA.Obtained form the National Institute of Biotechnology Information (NCBI)Open Reading Frame finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)

The data indicate that the PCM1 gene is composed of 37 exons spread overan area of approximately 92 kb of genomic sequence and cover perfectlythe whole 6075 nt of the PCM1 open reading frame.

Furthermore, Nucleotide Identify X (NIX,http://menu.hgmp.mrc.ac.uk/menu-bin/Nix/Nix.pl) analysis of 20 kb ofgenomic sequence upstream of the translation initiation codon identifieda 868 nt putative CpG island (67% GC) encompassing part of the 5′ end ofthe PCM1 transcript. Blast 2.0 homology search revealed perfect match ofpart of this CpG island with a CpG island isolated by the Sanger Centre[Cross, 1994, Nature Genetics 6, 236-244). Potential promoter sites werealso predicted around this CpG island. A potential consensuspolyadenylation signal is present 63 nt downstream of the stop codon.Exon 37gtttttcagAAACGGTGGGAGCCCAGAGTATATGAGATGTCTTCAGAGGCTCATCTAACTCTGTCCTTACATACTCAATGCATATATGAAAACAATACTAAATAAACATCTGATCTGTATAAAAATGTAA

PCM1 is represented by several ESTs in the Unigene database(http://www.ncbi.nlm.nih.gov/UniGene) and has been assigned the Unigeneidentification number Hs.75737. Expression data available in Unigeneshow that PCM1 is expressed in a wide variety of tissues (adrenal gland,aorta, bone, brain, breast, CNS, colon, eye, germ cell, head and neck,heart, kidney, lung, lymph, muscle, ovary, pancreas, parathyroid,placenta, prostate, smooth muscle, spleen, stomach, testis, thyroid,tonsil, uterus, whole embryo), including brain.

The open reading frame of PCM1 encodes a putative protein of 2024 aminoacids. A consensus nucleotide-binding site extending from amino acids1167-1174 was found. This amino acid stretch of ARILSGKT corresponds tothe consenus ATP/GTP binding motif that has been identified in severalATPases, kinases and GTP-binding proteins. In addition, it has thepotential of forming coiled-coils.

EXAMPLE 3 Analysis of PCM1 DNA Variation by Automated Bi-directional DNASequencing of Amplified Exons

For this analysis, 19 schizophrenia cases from the sample used in theassociation study together with 2 cases from each British multiplyaffected schizophrenia family that showed positive lods on chromosome8p21-22 (26 affected individuals) and 10 unrelated healthy controls wereused. In order to increase the chances of detecting a mutation inlinkage disequilibrium with schizophrenia, affected individuals thatcarried the alleles with higher frequencies in the cases than in thecontrols in the allelic association study that was described in theprevious chapter for the microsatellite markers D8S261, D8S2616 andD8S2615 were chosen.

PCM1 consists of 37 exons spanning about 92 kb of genomic sequence. Eachexon is however relatively small ranging in size from about 60 to 360bp. Marker D8S2616 lies within the intronic sequence of PCM1 betweenexons 4 and 5, while D8S261 is between exons 19 and 20.

The strategy chosen for mutation and DNA variation screening wasautomated bi-directional sequencing of PCR amplified exons. The wholeexonic sequence as well as more than 100 bp of intronic sequence oneither side of each exon were PCR amplified using M13 tailed primers asdescribed. The PCR product was then sequenced using the Thermo Sequenasekit. Each sequencing reaction contains two different M13 primerslabelled with different IR dyes (IRD700 and IRD800) in order to obtainsequence from both DNA strands of the PCR product (bi-directionalsequencing). In this way, both strands of the PCR products can becompletely sequenced. This approach is particularly useful for mutationdetection as both forward and reverse strands are assessedsimultaneously for the presence of a variation. In addition, detectionof heterozygotes is highly accurate. Furthermore, this sequence-basedmethod can detect the specific location of each mutation. Finally, theuse of the same sequencing primers for all PCR products makesbi-directional tailed primer sequencing economically feasible.Therefore, direct sequencing on the LI-COR was preferred to acombination of SSCP and sequencing as a more reliable and fasterapproach.

LI-COR's dual-dye automated DNA sequencer (Model 4200 IR² System) wasused to electrophorese and analyse the forward and reverse sequences ofthe bi-directional sequencing reactions in parallel. Sequence data wereinterpreted using the automated base-calling algorithms of LI-COR's BaseImagIR™ software.

Automatic alignment was performed by LI-COR's AlignIR™ software. Thesoftware aligns the sequences for the forward and reverse strand fromall the samples and produces a consensus. The consensus was generated bya minimum method that considers all base letters and generates an IUPACambiguity code for combination of bases at that position. The consensussequence is shown in a row below the last sample sequence while anannotation row below makes it easy to see ambiguities and mismatches inthe consensus sequence. When an ambiguity or a mismatch was encounteredat a certain position, windows containing the chromatogram files (.scffiles) for both forward and reverse strands for all the samples could beopened in order to assess the validity of the base calls. Wrong basecalls were edited in the AlignIR window by just highlighting thesequence and typing the appropriate letter or the IUPAC ambiguity code.

Heterozygous sites were identified by scanning the assembled sequencetraces for: (i) the presence of a drop in fluorescence peak height at aposition when compared to the respective peak height for all individualsthat are homozygous at the position and (ii) presence of another base (asecond peak) that accompanies the drop in fluorescence peak height.

Further polymorphisms and variants have also been identified as follows:

-   -   A to G SNP at base number 75915 of clone AB020866    -   T to C SNP at base number 79215 of clone AB020866    -   G to A SNP at base number 80123 of clone AB020866    -   C to T SNP at base number 87366 of clone AB020866    -   G to A SNP at base number 96961 of clone AB020866    -   G to A SNP at base number 97759 of clone AB020866    -   A to C SNP at base number 510 of clone AB020867    -   C to T SNP at base number 2450 of clone AB020867    -   Insertion/Deletion CC to C at base number 10153 of clone        AB020867    -   A to G SNP at base number 30364 of clone AB020867    -   T to C SNP at base number 32005 of clone AB020867    -   C to T SNP at base number 61847 of clone AB020867    -   A to C SNP at base number 51659 of clone AB020867    -   T to C SNP at base number 78722 of clone AB020867    -   T to C SNP at base number 75925 of clone AB020866    -   A to G SNP at base number 79214 of clone AB020866    -   A to G SNP at base number 80254 of clone AB020866    -   G to A SNP at base number 87507 of clone AB020866    -   A to C SNP at base number 61873 of clone AB020867    -   In/Del at base number 51745 of clone AB020867

It will be understood that the invention encompasses also these markersand those in linkage disequilibrium with them.

EXAMPLE 4 Analysis of Exon 4 and its Exon/Intron Boundaries

Exon 4 was screened first for mutations and DNA variation because markerD8S2616 that showed evidence of allelic association with schizophrenialies within the intronic sequence of PCM1 between exons 4 and 5.

A sequence of 507 bp was PCR amplified using M13 tailed primers. Thesequence of the primers has as follows:5′-GGATAACAATTTCACACAGG-CCAAGTGTCTTTGGTTATCTTCG-3′    M13 reverse primer(−21) forward PCR primer 5′-CACGACGTTGTAAAACGAC-AGTCCGAACATCCTCCTCCT-3′   M13 forward primer (−29) reverse PCR primer

This product included the whole of exon 4 (170 bp) while the rest of theamplified sequence was intronic sequence from both sides of this exon.PCR products were subsequently sequenced bi-directionally. Sequence wasobtained for all 10 controls and 45 cases.

Sequence analysis by LI-COR's AlignIR™ software followed. A singlenucleotide polymorphism was identified in the intronic sequence that was3′ to the exon. This is an A to G substitution that occurred in bothpatients and controls. Overall, 6 GG homozygotes, 1 AA homozygote and 3heterozygotes were identified in the samples from the controlindividuals while 23 GG homozygotes, 3 AA, homozygotes and 19heterozygotes were identified in the individuals with schizophrenia. TheDNA variation occurs at nucleotide position 80254 in the AB020866genomic clone. The AlignIR report that contains the novel DNA variantidentified in PCM1 using sequence analysis is presented in FIG. 2.

A candidate single nucleotide polymorphism was identified in the codingsequence at position 1100 of the PCM1 mRNA (NM_(—)006197) and 80123 ofthe AB020866 clone. It is a G to A substitution with relatively lowfrequency as only GG homozygotes and a few heterozygotes were observed.More specifically, 7 heterozygotes were observed among the cases whilethere were no heterozygotes in the controls. The rest of the individualswere GG homozygotes. This is a very interesting polymorphisms as itseems to produce an amino acid change in the protein sequence fromalanine (gct) to threonine (act). More individuals will have to beexamined in order to validate this polymorphism. The AlignIR report thatcontains this DNA variant is presented on FIG. 3.

The sequence of clone AB020866 was the reference genomic sequenceobtained from the Entrez nucleotide database and can be seen at thefirst line (ex4gnm) of FIG. 2. A variation occurs at nucleotide position80254 of the AB020866 clone. The consensus sequence obtained fromAlignIR can be seen at the last line (Consensus). Heterozygotes, aremarked R, G homozygotes are marked G and A homozygotes are marked A]

EXAMPLE 5 Analysis of Exon 5 and its Exon/Intron Boundaries

Exon 5 was screened for mutations and DNA variation at this stagebecause marker D8S2616 that showed evidence of allelic association withschizophrenia lies within the intronic sequence of PCM1 between exons 4and 5.

A sequence of 595 bp was PCR amplified using M13 tailed primers. Thesequence of the primers has as follows:5′-GGATAACAATTTCACACAGG-TGAGCCATTGATTATG-3′      M13 reverse primer(−21) forward PCR primer 5′-CACGACGTTGTAAAACGAC-AGTTGTCCCTGCAACCT-3′     M13 forward primer (−29) reverse PCR primer

This product included the whole of exon 5 (180 bp) while the rest of theamplified sequence was intronic sequence from both sides of this exon.PCR products were subsequently sequenced bi-directionally. Sequence wasobtained for 5 controls and 42 cases.

Sequence analysis by LI-COR's AlignIR™ software followed. Two singlenucleotide polymorphisms were identified in the intronic sequence thatwas 5′ to exon 5. One is a C to T substitution occurring 171 bp from thestart of exon 5 at position 87366 of the AB020866 genomic clone. In theindividuals examined, 1 TT homozygote and 5 heterozygotes wereidentified among the cases while the rest were CC homozygotes includingall 5 controls. The AlignIR report that contains this DNA variant ispresented on FIG. 4.

A second single nucleotide polymorphism which is a G to A substitutionoccurring 30 bp 5′ to the start of the exon at nucleotide position 87507of the AB020866 genomic clone. 2 GG homozygotes and 3 heterozygotes wereidentified in the controls examined while 11 GG homozygotes, 10 AAhomozygotes and 21 heterozygotes were identified in the cases. TheAlignIR report that contains this DNA variant is presented on FIG. 5.

All 4 single nucleotide polymorphisms identified in and around exons 4and 5 of the PCM1 were examined for possible alteration in a restrictionenzyme cutting site but none seemed to produce or obliterate one.

Discussion

PCM1 is part of the centrosome assembly. Although, possible roles in themaintenance of centrosome integrity and the regulation of themicrotubule cytoskeleton have been mentioned its function inneuropsychiatric disorders has not previously been elucidated. Itsprotein structure bears similarities to myosins, structural proteins andproteins involved in motility and/or transport (microtubule bindingproteins). As is described by Millar et al. [2000] many such structuralproteins are implicated in processes such as axon guidance,synaptogenesis, functioning of the synapse and intracellular transportalong axons and dendrites. It is now the case that PCM1 has a similarrole implicating this protein in the development of the nervous systemand/or neuronal activity and is therefore involved in the aetiology ofschizophrenia, PCM1 disorders and neuropsychiatric disorders.

Furthermore, PCM1 interacts with a brain-specific protein,huntingtin-associated protein 1 (HAP1). HAP1 binds to huntingtin in aglutamine repeat length-dependent manner as well as interacts withcytoskeletal, vesicular and motor proteins. In this way, it acts as anadaptor protein in order to mediate interactions among all thesedifferent molecules [Engelender, 1997, Hum Mol Genet 6: 2205-2212].

The fact that PCM1 interacts with proteins that are directly involvedwith neuronal function indicates that it may be involved in wide networkof proteins responsible for the normal functioning of neuronal cells.

In order to identify mutations as well as DNA sequence variation themethod of fluorescence bi-directional DNA sequencing was employed. DNAsequencing is the most sensitive method for finding DNA polymorphismsand mutations. In addition to its sensitivity, full DNA sequenceanalysis provides complete knowledge of the type, position and contextof every variation, regardless of whether it is a single nucleotidesubstitution or an insertion/deletion variation.

So far, two of the exons, exon 4 and exon 5 together with at least 100bp of intronic sequence on either side of each exon have been screenedfor single nucleotide polymorphisms. These exons were screened firstbecause marker D8S2616 that showed evidence for allelic association withschizophrenia lies in the intronic sequence between these two exons.

In exon 4, a G to A transition was identified at the 3′ intronicsequence of this exon in both patients and controls. It seems to be afrequent variant as homozygotes for each of the alternative alleles arepresent in the individuals screened as well as a number of heterozygotescontaining these alternative alleles. In addition, a single nucleotidepolymorphisms was identified in exon 4. It is again a G to A transitionbut the relative frequency of this variant is low because it wasidentified by infrequent heterozygotes among homozygotes. Thispolymorphism was identified as heterozygotes amongst individualsidentified as being schizophrenic and was not found in normal controls.It produces an amino acid change, from an alanine to a threonine, in thepredicted protein sequence of pericentriolar material 1. Such a muationshas been found to be associated with diseases in at least 140 otherinstances.

Two single nucleotide polymorphisms were identified in the intronicsequence 5′ to exon 5. Both substitutions are transitions, one from a Cto a T and the other from a G to an A and are 140 bp apart.

Although only a small part of the genomic sequence of PCM1 has beenscreened so far, the results are consistent with other studies of humanDNA sequence variation, which have identified on average 1 variable siteevery 217 bp [Halushka, 1999, Nat Genet 22: 239-247]. Furthermore, allthe nucleotide substitutions identified in this study were transitions,which is in agreement with other studies [Halushka, 1999, ref as above].Especially, A/G substitutions have the highest prevalence amongfunctional genes [Gojobori, 1982, J Mol Evol 18, 414-423]. Furtherscreening of the PCM1 regulatory and coding sequence are likely to yieldother types of mutations causing schizophrenia and otherneuropsychiatric disorders and PCM1 disorders. In the light of thepresent disclosure anyone skilled in the art of sequencing will findsuch mutations in increasing numbers of patients.

1. A method for determining the susceptibility of an individual to aneuropsychiatric disorder, or a method of diagnosis or prognosis of theneuropsychiatric disorder, the method comprising use of a marker locatedin the chromosomal region 8p21-22.
 2. A method as claimed in claim 1wherein the disorder is schizophrenia.
 3. A method as claimed in claim 1or claim 2 wherein the method comprises: (i) obtaining a nucleic acid orprotein sample from the individual; (ii) determining the structure,level of expression, and\or activity of the polypeptide encoded by thepericentriolar material 1 (PCM1) gene.
 4. A method as claimed in claim 1or claim 2 wherein the method comprises: (i)obtaining a sample ofnucleic acid from the individual, and (ii)determining in that sample,the presence or absence of a pericentriolar material 1 (PCM 1) marker.5. A method as claimed in claim 4 wherein the nucleic acid is RNA, cDNAor genomic DNA.
 6. A method as claimed in claim 4 or claim 5 wherein thePCM1 marker is selected from the group consisting of the any of thefollowing microsatellite repeats present in the NT_(—)000501 contig onchromosome 8p21.3-22: D8S2612; D8S2613; D8S2614; D8S2615; D8S2616;D8S2617; D8S2618, or D8S261, or a polymorphic marker which is in linkagedisequilibrium with any of these.
 7. A method as claimed in claim 5wherein the PCM1 marker is: D8S261; D8S2615; or D8S2616;
 8. A method asclaimed in any one of claims 4 to 7 wherein the PCM1 marker is withinthe PCM1 gene.
 9. A method as claimed in claim 8 wherein the PCM1 markeris an in the intronic sequence 3′ to exon 4; in exon 4; in the intronicsequence 5′ of exon 5 wherein said exon-intron are set out in FIG. 6.10. A method as claimed in claim 9 wherein the PCM1 marker is an SNPselected from the group consisting of the following positions numberedin accordance with the sequence of clone AB020866 as set out FIGS. 2-5:(i) 80254, (ii) 80123 (iii) 87366 (iv) 87507 or a polymorphic markerwhich is in linkage disequilibrium with any of these.
 11. A method asclaimed in claim 10 wherein the identity of the nucleotide at the SNP isshown at the corresponding numbered position in any one of FIGS. 2-5.12. A method as claimed in claims 10 or 11 wherein two or more of saidPCM 1 marker SNPs are assessed.
 13. A method as claimed in any of claims4 to 12 wherein the 8p21-22 region in assessed by determining thebinding of an oligonucleotide probe to the nucleic acid sample underconditions favourable for the specific annealing of these reagents totheir complementary sequences.
 14. A method as claimed in claim 13wherein the probe comprises all or part of (i) the PCM1 sequence shownin any one of FIGS. 1-5, or (ii) a polymorphic form of the PCM1 sequenceshown in any one of FIGS. 1-5, or (iii) the complement of either.
 15. Amethod as claimed in claim 13 or claim 14 wherein the probe comprises anucleic acid sequence which binds under stringent conditionsspecifically to one particular allele of a PCM1 marker and does not bindspecifically to other alleles of the PCM 1 marker.
 16. A method asclaimed in any one of claims 13 to 15 wherein the probe is labelled andbinding of the probe is determined by presence of the label.
 17. Amethod as claimed in any of claims 4 to 16 wherein the method comprisesamplifying a region of the 8p21-22 region comprising at least one PCM1marker.
 18. A method as claimed in claim 17 wherein the PCM1 gene isamplified.
 19. A method as claimed in claim 17 wherein the region of thePCM1 gene which is amplified is within the intronic sequence 3′ to exon4; in exon 4; or in the intronic sequence 5′ of exon
 5. 20. A method asclaimed in any one of claims 17 to 19 wherein a region of the 8p-22region is amplified by use of two oligonucleotide primers.
 21. A methodas claimed in any one of claims 4 to 12 wherein the PCM 1 marker isassessed by a method selected from the group consisting of: strandconformation polymorphic marker analysis; heteroduplex analysis; RFLPanalysis.
 22. A method as claimed in claim 3 wherein the level of mRNAexpression of the PCM1 gene is determined by Northern analysis orreverse transcriptase PCR.
 23. A method of determining the presence orabsence in a test sample of a PCM1 marker which is an SNP selected fromthe group consisting of the following positions numbered in accordancewith the sequence of clone AB020866 as set out FIGS. 2-5: (i) 80254,(ii) 80123, (iii) 87366, (iv) 87507, which method comprises determiningthe binding of an oligonucleotide probe to the nucleic acid sample,wherein the probe comprises all or part of (i) the PCM1 sequence shownin any one of FIGS. 2-5, or (ii) a polymorphic form of the PCM1 sequenceshown in any one of FIGS. 2-5, or (iii) the complement of either.
 24. Amethod of determining the presence or absence in a test sample of a PCM1marker which is an SNP selected from the group consisting of thefollowing positions numbered in accordance with the sequence of cloneAB020866 as set out FIGS. 2-5: (i) 80254, (ii) 80123, (iii) 87366, (iv)87507, which method comprises use of two oligonucleotide primers capableof amplifying a portion of the PCM1 sequence which portion comprises atleast one of said SNPs.
 25. A method as claimed in any one of thepreceding claims 4 to 21 wherein the PCM1 marker is assessed orconfirmed by nucleotide sequencing.
 26. An oligonucleotide probe for usein a method of any one of claims 13 to 16 or claim 23, which includes aPCM1 marker which is a microsatellite repeat present in the NT_(—)000501contig on chromosome 8p21.3-22: D8S2612; D8S2613; D8S2614; D8S2615;D8S2616; D8S2617; or D8S2618, or which an SNP selected from the groupconsisting of the following positions numbered in accordance with thesequence of clone AB020866 as set out FIGS. 2-5: (i) 80254, (ii) 80123,(iii) 87366, (iv)
 87507. 27. An oligonucleotide probe as claimed inclaim 26 which probe comprises all or part of (i) the PCM1 sequenceshown in any one of FIGS. 2-5, or (ii) a polymorphic form of the PCM1sequence shown in any one of FIGS. 2-5, or (iii) the complement ofeither,
 28. A PCR primer pair for use in a method of any one of claims17 to 20 or claim 24 which primer pair comprises first and secondprimers which hybridise to DNA in regions including or flanking the PCM1marker.
 29. A primer pair as claimed in claim 28 wherein at least one ofsaid primers is selected from the list consisting of: (SEQ ID NO: 102)5′-GGATAACAATTTCACACAGG-TGAGCCATTGATTATG-3′ (SEQ ID NO: 103)5′-CACGACGTTGTAAAACGAC-AGTTGTCCCTGCAACCT-3′ (SEQ ID NO: 100)5′-GGATAACAATTTCACACAGG-CCAAGTGTCTTTGGTTATCTTCG-3′ (SEQ ID NO: 101)5′-CACGACGTTGTAAAACGAC-AGTCCGAACATCCTCCTCCT-3′


30. A primer pair as claimed in claim 28 wherein at least one of saidprimers is selected from the list consisting of: 5′AAT TCC CCA AAC AAAACA ACA3′ (SEQ ID NO: 85) 5′AGG CTA TCC TTT CCT CAG CA3′ (SEQ ID NO: 86)5′ATG TTC AGC CAC CAT CGT CT3′ (SEQ ID NO: 87) 5′CAG TGT CGC TGG AAA GTTGA3′ (SEQ ID NO: 88) 5′TCC CGA AGT GCT AGG ATT ACA3′ (SEQ ID NO: 89)5′GCT CAG CAG GAA GAG GAA TG3′ (SEQ ID NO: 90) 5′AGA GGC CAG GCA CAA AAGTA3′ (SEQ ID NO: 91) 5′AAC ATT CCA GCA TCC CAA AG3′ (SEQ ID NO: 92)5′GAC CCA CTG CCA CAC TCT TT3′ (SEQ ID NO: 93) 5′GGA GTG CGG CAT GAA ATTAT3′ (SEQ ID NO: 94) 5′ATA TGT ATA CAA TGT GTA TCT GTA (SEQ ID NO: 95)TC3′ 5′CCT TTT AGT TCC CAT TCC CAT T3′ (SEQ ID NO: 96) 5′TGA TGC AGG AGAATT GCT TG3′ (SEQ ID NO: 97) 5′CCT ACT TGG CTG GGA TTC TG3′ (SEQ ID NO:98)


31. A kit for determining the susceptibility of an individual toschizophrenia, or a method of diagnosis or prognosis of schizophrenia,the kit comprising a probe and\or primer of any one of claims 26 to 30.32. A method of schizophrenia therapy, which method including the stepof screening an individual for a genetic predisposition to schizophreniain accordance with the method of any one of claims 1 to 22, whereby thepredisposition is correlated with a PCM1 marker, and if a predispositionis identified, providing therapeutic treatment for the individual.
 33. Amethod for identifying or isolating genetic loci associated withsusceptibility to schizophrenia comprising screening genomic librarieswith genetic sequence derived from PCM1 polymorphic markers located inthe chromosomal region 8p21.3 and identifying open reading frames inregions adjacent to said genetic sequence.
 34. A method for mappingpolymorphic markers which are associated with susceptibility toschizophrenia, the method comprising identifying polymorphic markerswhich are in linkage disequilibrium with a PCM1 marker which is amicrosatellite repeat present in the NT_(—)000501 contig on chromosome8p21.3-22: D8S261; D8S2612; D8S2613; D8S2614; D8S2615; D8S2616; D8S2617;or D8S2618, or which an SNP selected from the group consisting of thefollowing positions numbered in accordance with the sequence of cloneAB020866 as set out FIGS. 2-5: (i) 80254, (ii) 80123, (iii) 87366, (iv)87507.
 35. A method of identifying a molecule for use in the diagnosis,prognosis or treatment of schizophrenia, which method comprises:admixing a test substance with a polypeptide encoded by a nucleic acidmolecule comprising the PCM1 gene or a gene located within 1000 kb ofthe PCM1 locus and in linkage disequilibrium therewith; and measuringthe level of activity of the polypeptide.
 36. A method of identifying amolecule for use in the diagnosis, prognosis or treatment ofschizophrenia, which method comprises: admixing a test substance with apolypeptide encoded by a nucleic acid molecule comprising the PCM1 geneor a gene located within 1000 kb of the PCM1 locus and in linkagedisequilibrium therewith; and determining the binding of the testsubstance to the polypeptide.
 37. An antibody specific for a polypeptideencoded by the PCM1 gene for use as a diagnostic and prognostic forschizophrenia.
 38. A nucleic acid molecule comprising the PCM1 gene or agene located within 1000 kb of the PCM1 locus and in linkagedisequilibrium therewith for use in the treatment of schizophrenia. 39.A polypeptide encoded by the PCM1 gene or a gene located within 1000 kbof the PCM1 locus and in linkage disequilibrium therewith for use in thetreatment of schizophrenia.
 40. A method of treatment of schizophreniacomprising administering to a patient a substance which modulatesexpression from the PCM1 gene or a gene located within 1000 kb of thePCM1 locus and in linkage disequilibrium therewith, or administering acompound which modulates the level of activity of the PCM1 gene productor a gene located within 1000 kb of the PMC1 locus and in linkagedisequilibrium therewith.